Massively multiplexed nucleic acid detection with Cas13.

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

CRISPR/Cas Technology: The Unique Synthetic Biology Genome-Editing Tool Shifting the Paradigm in Viral Diagnostics, Defense, and Therapeutics.

The emergence of the COVID-19 pandemic has starkly exposed our significantly limited ability to promptly identify and respond to emergent biological threats. Consequently, there is an urgent need to advance biotechnological methods for addressing both known and unforeseen biological hazards. Recently, the CRISPR/Cas system has revolutionized genetic engineering, enabling precise and efficient synthetic biology applications. Therefore, this review aims to provide a comprehensive introduction to the fundamental principles underlying the CRISPR/Cas system and assess the advantages and limitations of various CRISPR/Cas-based techniques applicable to the detection of, defense against, and treatment of viral infections. These techniques include viral diagnostics, the development of antiviral vaccines, B cell engineering for antibody production, viral activation/interference, and epigenetic modifications. Furthermore, this review delves into the challenges and bioethical considerations associated with use of the CRISPR/Cas system. With the continuous evolution of technology, the CRISPR/Cas system holds considerable promise for addressing both existing and unforeseen biological threats.

Electronic Health Record-Based Algorithm for Monitoring Respiratory Virus-Like Illness.

Viral respiratory illness surveillance has traditionally focused on single pathogens (e.g., influenza) and required fever to identify influenza-like illness (ILI). We developed an automated system applying both laboratory test and syndrome criteria to electronic health records from 3 practice groups in Massachusetts, USA, to monitor trends in respiratory viral-like illness (RAVIOLI) across multiple pathogens. We identified RAVIOLI syndrome using diagnosis codes associated with respiratory viral testing or positive respiratory viral assays or fever. After retrospectively applying RAVIOLI criteria to electronic health records, we observed annual winter peaks during 2015-2019, predominantly caused by influenza, followed by cyclic peaks corresponding to SARS-CoV-2 surges during 2020-2024, spikes in RSV in mid-2021 and late 2022, and recrudescent influenza in late 2022 and 2023. RAVIOLI rates were higher and fluctuations more pronounced compared with traditional ILI surveillance. RAVIOLI broadens the scope, granularity, sensitivity, and specificity of respiratory viral illness surveillance compared with traditional ILI surveillance.

Validation of the VirCapSeq-VERT system for differential diagnosis, detection, and surveillance of viral infections.

IMPORTANCE: Broad range assay for accurate and sensitive diagnostics.

Comparison of the performance of two targeted metagenomic virus capture probe-based methods using reference control materials and clinical samples.

Viral enrichment by probe hybridization has been reported to significantly increase the sensitivity of viral metagenomics. This study compares the analytical performance of two targeted metagenomic virus capture probe-based methods: (i) SeqCap EZ HyperCap by Roche (ViroCap) and (ii) Twist Comprehensive Viral Research Panel workflow, for diagnostic use. Sensitivity, specificity, and limit of detection were analyzed using 25 synthetic viral sequences spiked in increasing proportions of human background DNA, eight clinical samples, and American Type Culture Collection (ATCC) Virome Virus Mix. Sensitivity and specificity were 95% and higher for both methods using the synthetic and reference controls as gold standard. Combining thresholds for viral sequence read counts and genome coverage [respectively 500 reads per million (RPM) and 10% coverage] resulted in optimal prediction of true positive results. Limits of detection were approximately 50-500 copies/mL for both methods as determined by ddPCR. Increasing proportions of spike-in cell-free human background sequences up to 99.999% (50 ng/mL) did not negatively affect viral detection, suggesting effective capture of viral sequences. These data show analytical performances in ranges applicable to clinical samples, for both probe hybridization metagenomic approaches. This study supports further steps toward more widespread use of viral metagenomics for pathogen detection, in clinical and surveillance settings using low biomass samples. IMPORTANCE: Viral metagenomics has been gradually applied for broad-spectrum pathogen detection of infectious diseases, surveillance of emerging diseases, and pathogen discovery. Viral enrichment by probe hybridization methods has been reported to significantly increase the sensitivity of viral metagenomics. During the past years, a specific hybridization panel distributed by Roche has been adopted in a broad range of different clinical and zoonotic settings. Recently, Twist Bioscience has released a new hybridization panel targeting human and animal viruses. This is the first report comparing the performance of viral metagenomic hybridization panels.

Practical updates in clinical antiviral resistance testing.

The laboratory diagnosis of antiviral resistance is a quickly changing field due to new drug availability, the sunsetting of older drugs, the development of novel technologies, rapid viral evolution, and the financial/logistic pressures of the clinical laboratory. This mini-review summarizes the current state of clinically available antiviral resistance testing in the United States in 2024, covering the most commonly used test methods, mechanisms, and clinical indications for herpes simplex virus, cytomegalovirus, human immunodeficiency virus, influenza, hepatitis B virus, and hepatitis C virus drug resistance testing. Common themes include the move away from phenotypic to genotypic methods for first-line clinical testing, as well as uncertainty surrounding the clinical meaningfulness of minority variant detection as next-generation sequencing methods have become more commonplace.

At-home testing for respiratory viruses: a minireview of the current landscape.

The landscape of at-home testing using over-the-counter (OTC) tests has been evolving over the last decade. The United States Food and Drug Administration Emergency Use Authorization rule has been in effect since the early 2000s, and it was widely employed during the severe acute respiratory syndrome coronavirus 2 pandemic to authorize antigen and nucleic acid detection tests for use in central laboratories as well as OTC. During the pandemic, the first at-home tests for respiratory viruses became available for consumer use, which opened the door for additional respiratory virus OTC tests. Concerns may exist regarding the public's ability to properly collect samples, perform testing, interpret results, and report results to public health authorities. However, favorable comparison studies between OTC testing and centralized laboratory test results suggest that OTC testing may have a place in healthcare, and it is likely here to stay. This mini-review of OTC tests for viral respiratory diseases will briefly cover the regulatory and reimbursement environment, current OTC test availability, as well as the advantages and limitations of OTC tests.

bvnGPS: a generalizable diagnostic model for acute bacterial and viral infection using integrative host transcriptomics and pretrained neural networks.

MOTIVATION: The confusion of acute inflammation infected by virus and bacteria or noninfectious inflammation will lead to missing the best therapy occasion resulting in poor prognoses. The diagnostic model based on host gene expression has been widely used to diagnose acute infections, but the clinical usage was hindered by the capability across different samples and cohorts due to the small sample size for signature training and discovery. RESULTS: Here, we construct a large-scale dataset integrating multiple host transcriptomic data and analyze it using a sophisticated strategy which removes batch effect and extracts the common information from different cohorts based on the relative expression alteration of gene pairs. We assemble 2680 samples across 16 cohorts and separately build gene pair signature (GPS) for bacterial, viral, and noninfected patients. The three GPSs are further assembled into an antibiotic decision model (bacterial-viral-noninfected GPS, bvnGPS) using multiclass neural networks, which is able to determine whether a patient is bacterial infected, viral infected, or noninfected. bvnGPS can distinguish bacterial infection with area under the receiver operating characteristic curve (AUC) of 0.953 (95% confidence interval, 0.948-0.958) and viral infection with AUC of 0.956 (0.951-0.961) in the test set (N = 760). In the validation set (N = 147), bvnGPS also shows strong performance by attaining an AUC of 0.988 (0.978-0.998) on bacterial-versus-other and an AUC of 0.994 (0.984-1.000) on viral-versus-other. bvnGPS has the potential to be used in clinical practice and the proposed procedure provides insight into data integration, feature selection and multiclass classification for host transcriptomics data. AVAILABILITY AND IMPLEMENTATION: The codes implementing bvnGPS are available at https://github.com/Ritchiegit/bvnGPS. The construction of iPAGE algorithm and the training of neural network was conducted on Python 3.7 with Scikit-learn 0.24.1 and PyTorch 1.7. The visualization of the results was implemented on R 4.2, Python 3.7, and Matplotlib 3.3.4.

Simple, sensitive, and visual detection of 12 respiratory pathogens with one-pot-RPA-CRISPR/Cas12a assay.

Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 100 copies/µL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.

Epidemiological study of post-pandemic pediatric common respiratory pathogens using multiplex detection.

BACKGROUND: The burden and characteristics of respiratory viral infections in children hospitalized for acute respiratory tract infections (ARTIs) during the post-COVID-19 pandemic era are unclear. We analyzed the epidemiological and clinical characteristics of pediatric patients hospitalized with common respiratory virus infections before and after relaxation of non-pharmaceutical interventions in Hangzhou, China and evaluated the diagnostic value of the six-panel respiratory pathogen detection system. METHODS: Six types of respiratory viruses were detected in respiratory samples from children with suspected ARTIs by multiplex real-time quantitative polymerase chain reaction (RT-qPCR). Changes in virus detection rates and epidemiological and clinical characteristics, obtained from electronic health records, were analyzed. Binary logistic regression was used to identify respiratory tract infections risk factors. Multiplex RT-qPCR and targeted next-generation sequencing results were compared in random samples. RESULTS: Among the 11,056 pediatric samples, 3228 tested positive for one or more of six common respiratory pathogens. RSV and PIV-3 detection rates differed significantly across age groups (both P < 0.001), and were more common in younger children. PIV-1 was more common in infants, toddlers, and preschoolers than in school-age children (P < 0.001). FluB was predominantly detected in school-age children (P < 0.001). RSV-, ADV-, and PIV-1-positivity rates were higher in 2022 than in 2023. Seasonal viral patterns differed across years. RSV (OR 9.156. 95% CI 5.905-14.195) and PIV-3 (OR 1.683, 95% CI 1.133-2.501) were risk factors for lower respiratory tract infections. RSV-positivity was associated with severe pneumonia (P = 0.044). PIV-3 (OR 0.391, 95% CI 0.170-0.899), summer season (OR 1.982, 95% CI 1.117-3.519), and younger age (OR 0.938, 95% CI 0.893-0.986) influenced pneumonia severity. Multiplex RT-qPCR showed good diagnostic performance. CONCLUSION: After changes in COVID-19 prevention and control strategies, six common respiratory viruses in children were prevalent in 2022-2023, with different seasonal epidemic characteristics and age proclivities. RSV and PIV-3 cause lower, and FluA, FluB, and ADV more typically cause upper respiratory tract infections. Infancy and summer season influence severe pneumonia risk. Multiplex RT-qPCR is valuable for accurate and timely detection of respiratory viruses in children, which facilitates management, treatment, and prevention of ARTIs.

Integrated, Multi-cohort Analysis Identifies Conserved Transcriptional Signatures across Multiple Respiratory Viruses.

Respiratory viral infections are a significant burden to healthcare worldwide. Many whole genome expression profiles have identified different respiratory viral infection signatures, but these have not translated to clinical practice. Here, we performed two integrated, multi-cohort analyses of publicly available transcriptional data of viral infections. First, we identified a common host signature across different respiratory viral infections that could distinguish (1) individuals with viral infections from healthy controls and from those with bacterial infections, and (2) symptomatic from asymptomatic subjects prior to symptom onset in challenge studies. Second, we identified an influenza-specific host response signature that (1) could distinguish influenza-infected samples from those with bacterial and other respiratory viral infections, (2) was a diagnostic and prognostic marker in influenza-pneumonia patients and influenza challenge studies, and (3) was predictive of response to influenza vaccine. Our results have applications in the diagnosis, prognosis, and identification of drug targets in viral infections.

Limited availability of methods for the detection of xenotransplantation-relevant viruses in veterinary laboratories.

BACKGROUND: The German Xenotransplantation Consortium is in the process to prepare a clinical trial application (CTA) on xenotransplantation of genetically modified pig hearts. In the CTA documents to the central and national regulatory authorities, that is, the European Medicines Agency (EMA) and the Paul Ehrlich Institute (PEI), respectively, it is required to list the potential zoonotic or xenozoonotic porcine microorganisms including porcine viruses as well as to describe methods of detection in order to prevent their transmission. The donor animals should be tested using highly sensitive detection systems. I would like to define a detection system as the complex including the actual detection methods, either PCR-based, cell-based, or immunological methods and their sensitivity, as well as sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls. Lessons learned from the identification of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) in the xenotransplanted heart in the recipient in the Baltimore study underline how important such systems are. The question is whether veterinary laboratories can supply such assays. METHODS: A total of 35 veterinary laboratories in Germany were surveyed for their ability to test for selected xenotransplantation-relevant viruses, including PCMV/PRV, hepatitis E virus, and porcine endogenous retrovirus-C (PERV-C). As comparison, data from Swiss laboratories and a laboratory in the USA were analyzed. Furthermore, we assessed which viruses were screened for in clinical and preclinical trials performed until now and during screening of pig populations. RESULTS: Of the nine laboratories that provided viral diagnostics, none of these included all potential viruses of concern, indeed, the most important assays confirmed in recent human trials, antibody detection of PCMV/PRV and screening for PERV-C were not available at all. The situation was similar in Swiss and US laboratories. Different viruses have been tested for in first clinical and preclinical trials performed in various countries. CONCLUSION: Based on these results it is necessary to establish special virological laboratories able to test for all xenotransplantation-relevant viruses using validated assays, optimally in the xenotransplantation centers.

Advances in rapid point-of-care virus testing.

Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology, molecular biology and cancer research, and it is important to study the discovered viruses with potential applications. Therefore, the development of immediate and rapid viral detection methods for the prevention and treatment of viral diseases as well as the study of viruses has attracted extensive attention from scientists. With the continuous progress of science and technology, especially in the field of bioanalysis, a series of new detection techniques have been applied to the on-site rapid detection of viruses, which has become a powerful approach for human beings to fight against viruses. In this paper, the latest research progress of rapid point-of-care detection of viral nucleic acids, antigens and antibodies is presented. In addition, the advantages and disadvantages of these technologies are discussed from the perspective of practical application requirements. Finally, the problems and challenges faced by rapid viral detection methods and their development prospects are discussed.

A microfluidic-based quantitative analysis system for the multiplexed genetic diagnosis of human viral infections using colorimetric loop-mediated isothermal amplification.

In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 µL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.

Respiratory Viruses Co-detection on a Multiplex RT-PCR Panel: a Comparative Clinical Study.

BACKGROUND: The FilmArray Respiratory Panel RP 2.1 plus (FilmArray RP) is a point-of-care syndromic panel for respiratory pathogens. Although highly valuable in the clinical settings, the co-detection of pathogens in FilmArray RP may confound result interpretation. METHODS: Nasopharyngeal swab specimens collected from patients with respiratory symptoms were analyzed by comparing co-detection results from FilmArray RP with those of Allplex Respiratory Panels (Allplex RP: Power-Chek for SARS-CoV-2). RESULTS: Out of 765 FilmArray RP tests, 143 (18.7%) showed co-detections (two: 122 (85.3%), three: 18 (12.6%), four: 2 (1.4%), and five viruses: 1 (0.7%). The most frequent co-detection was human rhinovirus/enterovirus (HRV/HEV) with respiratory syncytial virus (RSV) (22.3%, 32/143). The overall discordance rate between Film-Array RP and other tests was 32.9%. Notably, discordance in detecting adenovirus (AdV) was significant, with cases detected by FilmArray often not appearing in Allplex RP. CONCLUSIONS: Discordances were varied by virus combination. It is advisable to perform additional confirmatory testing based on clinical relevance.

Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

Gold nanoparticles-based biosensors: pioneering solutions for bacterial and viral pathogen detection-a comprehensive review.

Gold Nanoparticles (AuNPs) have gained significant attention in biosensor development due to their unique physical, chemical, and optical properties. When incorporated into biosensors, AuNPs offer several advantages, including a high surface area-to-volume ratio, excellent biocompatibility, ease of functionalization, and tunable optical properties. These properties make them ideal for the detection of various biomolecules, including proteins, nucleic acids, and bacterial and viral biomarkers. Traditional methods for detecting bacteria and viruses, such as RT-PCR and ELISA, often suffer from complexities, time consumption, and labor intensiveness. Consequently, researchers are continuously exploring novel devices to address these limitations and effectively detect a diverse array of infectious pathogenic microorganisms. In light of these challenges, nanotechnology has been instrumental in refining the architecture and performance of biosensors. By leveraging advancements in nanomaterials and strategies of biosensor fabrication the sensitivity and specificity of biosensors can be enhanced, enabling more precise detection of pathogenic bacteria and viruses. This review explores the versatility of AuNPs in detecting a variety of biomolecules, including proteins, nucleic acids, and bacterial and viral biomarkers. Furthermore, it evaluates recent advancements in AuNPs-based biosensors for the detection of pathogens, utilizing techniques such as optical biosensors, lateral flow immunoassays, colorimetric immunosensors, electrochemical biosensors, and fluorescence nanobiosensors. Additionally, the study discusses the existing challenges in the field and proposes future directions to improve AuNPs-based biosensors, with a focus on enhancing sensitivity, selectivity, and their utility in clinical and diagnostic applications.

Rio Negro Virus Infection, Bolivia, 2021.

In May 2021, an agricultural worker originally from Trementinal, Argentina, sought treatment for febrile illness in Tarija, Bolivia, where he resided at the time of illness onset. The patient tested negative for hantavirus RNA, but next-generation sequencing of a serum sample yielded a complete genome for Rio Negro virus.

Sequence-based diagnostics and precision medicine in bacterial and viral infections: from bench to bedside.

PURPOSE OF REVIEW: Nucleic acid sequence-based organism identification plays an important role in the diagnosis and management of transplant and cancer-associated infectious diseases. Here, we provide a high-level overview of advanced sequencing technologies, discuss test performance, and highlight unmet research needs with a focus on immunocompromised hosts. RECENT FINDINGS: Next-generation sequencing (NGS) technologies are powerful tools with a growing role in managing immunocompromised patients with suspected infection. Targeted NGS (tNGS) can identify pathogens directly from patient specimens, especially for mixed samples, and has been used to detect resistance mutations in transplant-related viruses (e.g. CMV). Whole-genome sequencing (WGS) is increasingly used for outbreak investigations and infection control. Metagenomic NGS (mNGS) is useful for hypothesis-free testing and can simultaneously assess pathogens and host response to infection. SUMMARY: NGS testing increases diagnostic yield relative to standard culture and Sanger sequencing but may be limited by high cost, turnaround times, and detection of unexpected organisms or commensals of uncertain significance. Close collaboration with the clinical microbiology laboratory and infectious diseases is recommended when NGS testing is considered. Additional research is required to understand which immunocompromised patients are most likely to benefit from NGS testing, and when testing should ideally be performed.