This article, aimed at nonlaboratorians such as healthcare providers, public health professionals, and policymakers, provides basic concepts and terminology to enable better understanding of other manuscripts in this advanced molecular detection journal supplement. This article focuses on 3 aspects of advanced molecular detection: pathogen genomics, bioinformatics, and public health application, while providing additional resources for understanding.

Advanced molecular detection (AMD) refers to the integration of next-generation sequencing, epidemiologic, and bioinformatics data to drive public health actions. As new AMD technologies emerge, it is critical to ensure those methods are used in communities that are most affected by disease-induced illness and death. We describe strategies and opportunities for using AMD approaches to improve health in those communities, which include improving access to pathogen sequencing, increasing data linkages, and using pathogen sequencing for those diseases where sequencing technologies can provide the best health outcome. Such strategies can help address and prevent differences in health outcomes in various populations, such as rural and tribal communities, persons with underlying health issues, and other populations that experience higher risks for infectious disease.

The Next-Generation Sequencing (NGS) Quality Initiative addresses laboratory challenges faced when performing NGS by developing tools and resources to build a robust quality management system. Here, we illustrate how those products support laboratories in navigating complex regulatory environments and quality-related challenges while implementing NGS effectively in an evolving landscape.

Bioinformatic software containerization, the process of packaging software that encapsulates an application together with all necessary dependencies to simplify installation and use, has improved the deployment and management of next-generation sequencing workflows in both clinical and public health laboratories. Containers have increased next-generation sequencing workflow reproducibility and broadened their usage across different laboratories. We highlight the value of the State Public Health Bioinformatics community’s containerized software repository during the COVID-19 pandemic.

The role of genomics in outbreak response and pathogen surveillance has expanded and ushered in the age of pathogen intelligence. Genomic surveillance enables detection and monitoring of novel pathogens; case clusters; and markers of virulence, antimicrobial resistance, and immune escape. We can leverage pathogen genomic diversity to estimate total pathogen burden in populations and environments, which was previously challenging because of unreliable data. Pathogen genomics might allow pathogen burdens to be estimated by sequencing even a small percentage of cases. Deeper genomic epidemiology analyses require multidisciplinary collaboration to ensure accurate and actionable real-time pathogen intelligence.

Mitigating antimicrobial resistance (AMR) is a public health priority to preserve antimicrobial treatment options. The Washington State Department of Health in Washington, USA, piloted a process to leverage longitudinal genomic surveillance on the basis of whole-genome sequencing (WGS) and a genomics-first cluster definition to enhance AMR surveillance. Here, we outline the approach to collaborative surveillance and describe the pilot using 6 carbapenemase-producing organism outbreaks of 3 species: Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. We also highlight how we applied the approach to an emerging outbreak. We found that genomic and epidemiologic data define highly congruent outbreaks. By layering genomic and epidemiologic data, we refined linkage hypotheses and addressed gaps in traditional epidemiologic surveillance. With the accessibility of WGS, public health agencies must leverage new approaches to modernize surveillance for communicable diseases.

Airport-based pathogen monitoring is a critical tool that can contribute to early detection and characterization of existing and new pathogen threats. A novel public–private partnership between an airport spa group, a biotech company, and the Centers for Disease Control and Prevention was instrumental in establishing a multimodal pathogen genomic surveillance program at US international airports. That public–private partnership addressed critical challenges that neither party could overcome independently, resulting in the development and deployment of a scalable, flexible early warning system for pathogen detection and public health monitoring.

Genomic epidemiology offers insight into the transmission and evolution of respiratory viruses. We used metagenomic sequencing from negative SARS-CoV-2 rapid antigen tests to identify a wide range of respiratory viruses and generate full genome sequences. This process offers a streamlined mechanism for broad respiratory virus genomic surveillance.

The COVID-19 pandemic has been marked by continuous emergence of novel SARS-CoV-2 variants. Questions remain about the mechanisms with which those variants establish themselves in new geographic areas. We performed a discrete phylogeographic analysis on 18,529 sequences of the SARS-CoV-2 Omicron BA.5 sublineage sampled during February–June 2022 to elucidate emergence of that sublineage in different regions of the United States. The earliest BA.5 sublineage introductions came from Africa, the putative variant origin, but most were from Europe, matching a high volume of air travelers. In addition, we discovered extensive domestic transmission between different US regions, driven by population size and cross-country transmission between key hotspots. We found most BA.5 virus transmission within the United States occurred between 3 regions in the southwestern, southeastern, and northeastern parts of the country. Our results form a framework for analyzing emergence of novel SARS-CoV-2 variants and other pathogens in the United States.

We conducted retrospective analysis of the emergence of the SARS-CoV-2 JN.1 variant in US wastewater during November 2023–July 2024 using Aquascope, a bioinformatics pipeline for the National Wastewater Surveillance System. This study highlights the value of open-source bioinformatics tools in tracking pathogen variants for public health monitoring.

In the United States, SARS-CoV-2 genomic surveillance initially relied almost entirely on residual diagnostic specimens from nucleic acid amplification–based tests. However, use of those tests waned after the end of the COVID-19 Public Health Emergency on May 11, 2023. In Dane County, Wisconsin, we partnered with local- and state-level public health agencies and the South Central Library System to continue genomic surveillance by obtaining SARS-CoV-2 genome sequences from freely available community rapid antigen tests (RATs). During August 15, 2023–February 29, 2024, we received 227 RAT samples, from which we generated 127 sequences with >10× depth of coverage for >90% of the SARS-CoV-2 genome. In a subset of tests, lower cycle threshold values correlated with sequence success. Our results demonstrated that collecting and sequencing results from RATs in partnership with community sites is a practical approach for sustaining SARS-CoV-2 genomic surveillance.

Highly pathogenic avian influenza A(H5N1) virus has caused a multistate outbreak among US dairy cattle, spreading across 16 states and infecting hundreds of herds since its onset. We rapidly developed and optimized PCR-based detection assays and sequencing protocols to support H5N1 molecular surveillance. Using 214 retail milk samples from 20 states for methods development, we found that H5N1 virus concentrations by digital PCR strongly correlated with quantitative PCR cycle threshold values; digital PCR exhibited greater sensitivity. Metagenomic sequencing after hybrid selection was best for higher concentration samples, whereas amplicon sequencing performed best for lower concentrations. By establishing these methods, we were able to support the creation of a statewide surveillance program to perform monthly testing of bulk milk samples from all dairy cattle farms in Massachusetts, USA, which remain negative to date. The methods, workflow, and recommendations described provide a framework for others aiming to conduct H5N1 surveillance efforts.

A monkeypox virus genomic surveillance pilot began in King County, Washington, USA, during the 2022 outbreak. Genomic surveillance proved critical in determining local versus international exposure of a case where no known exposures were identified by interview, illustrating the value of genomics in case investigation and public health practice.

Detecting and responding to clusters of rapid HIV transmission is a core HIV prevention strategy in the United States, guiding public health interventions and identifying gaps in prevention and care services. In 2016, the Centers for Disease Control and Prevention (CDC) initiated molecular cluster detection using data from 27 jurisdictions. During 2016–2023, CDC expanded sequence reporting nationwide and deployed Secure HIV-TRACE, an application supporting health department (HD) molecular cluster detection. CDC conducts molecular cluster detection quarterly; state and local HDs analyze local data monthly. HDs began routinely reporting clusters to CDC by using cluster report forms in 2020. During 2018–2023, CDC identified 404 molecular clusters of rapid HIV transmission; 325 (80%) involved multiple jurisdictions. During 2020–2023, HDs reported 298 molecular clusters to CDC; 249 were first detected by HDs. Expanding molecular cluster detection has provided a foundation for improving service delivery to networks experiencing rapid HIV transmission.

We assessed turnaround times in the national Listeria monocytogenes genomic surveillance system in Australia before and after decentralized sequencing. Using 1,204 samples collected during 2016–2023, we observed statistically significant reductions in median time from sample collection to issuance of national genomic surveillance report to 26 days, despite sample numbers doubling in 2022 and 2023. During 2016–2018, all jurisdictions referred samples to the National Listeria Reference Laboratory for sequencing and analysis, but as jurisdictional sequencing capacity increased, 4 jurisdictions transitioned to sequencing their own samples and referring sequence data to the national laboratory. One jurisdiction had well-established genomics capacity, transitioned without noticeable disruption, and continued to improve. Another 3 jurisdictions initially had increased turnaround times, highlighting the need for defined sequence referral mechanisms. Overall, timeliness and throughput improved, and sequencing decentralization strengthened Australia’s genomic surveillance system while maintaining timeliness. The practices described could be beneficial and achievable in other countries.

We report the detection of a Shigella sonnei outbreak from a small investigation in the San Francisco Bay area, California, USA, in 2024. By combining outbreak investigation with genomic sequencing, we show the utility of phylodynamics to aid outbreak investigations of bacterial pathogens by state or local public health departments.

The Centers for Disease Control and Prevention’s Arctic Investigations Program evaluated whole-genome sequencing (WGS) workflows and bioinformatics pipelines developed by the Centers’ Streptococcus Laboratory. We compared WGS-based antimicrobial drug resistance predictions with phenotypic testing for group B (n = 130) and group A (n = 217) Streptococcus and Streptococcus pneumoniae (n = 293). Isolates were collected in Alaska during January 2019–February 2021. We also included a historical phenotypically nonsusceptible subset. Concordances between phenotypic testing and WGS predictions were 99.9% (895/896) for group B Streptococcus, 100% (1,298/1,298) for group A Streptococcus, and 99.98% (3,516/3,517) for S. pneumoniae. Common resistance determinants were ermTR, ermB, and mef for macrolides, tetM for tetracyclines, and gyrA and parC for levofloxacin. S. pneumoniae trimethoprim/sulfamethoxazole nonsusceptibility was associated with folP gene insertions and folA mutations. In 2022, the Arctic Investigations Program transitioned Streptococcus spp. workflows to WGS, enabling more rapid monitoring and prevention of invasive disease.

In the United States, Shiga toxin–producing Escherichia coli (STEC) outbreaks cause >265,000 infections and cost $280 million annually. We investigated REPEXH01, a persistent strain of STEC O157:H7 associated with multiple sources, including romaine lettuce and recreational water, that has caused multiple outbreaks since emerging in late 2015. By comparing the genomes of 729 REPEXH01 isolates with those of 2,027 other STEC O157:H7 isolates, we identified a highly conserved, single base pair deletion in espW that was strongly linked to REPEXH01 membership. The biological consequence of that deletion remains unclear; further studies are needed to elucidate its role in REPEXH01. Additional analyses revealed that REPEXH01 isolates belonged to Manning clade 8; possessed the toxins stx2a, stx2c, or both; were predicted to be resistant to several antimicrobial compounds; and possessed a diverse set of plasmids. Those factors underscore the need to continue monitoring REPEXH01 and clarify aspects contributing to its emergence and persistence.

Whole-genome sequencing (WGS) is routine for surveillance of Shiga toxin–producing Escherichia coli human isolates in France. Protocols use EnteroBase hierarchical clustering at <5 allelic differences (HC5) as screening for cluster detection. We assessed current implementation after 5 years for 1,002 sequenced isolates. From genomic distances of serotypes O26:H11, O157:H7, O80:H2, and O103:H2, we determined statistical thresholds for cluster determination and compared those with HC5 clusters. Thresholds varied by serotype, 5–16 allelic distances and 15–20 single-nucleotide polymorphisms, showing limits of a single-threshold approach. We confirmed validity of HC5 screening for 3 serotypes because statistical thresholds had limited effect on isolate clustering (high sensitivity and specificity). For O80:H2, results suggest that HC5 is less reliable, and other approaches should be explored. Public health officials should regularly assess WGS used for Shiga toxin–producing E. coli surveillance to account for serotype and genomic evolution and to interpret WGS-linked isolates in light of epidemiologic data.

Gene PCR and sequencing using 16S ribosomal RNA (rRNA) can help diagnose challenging bacterial infections. Data on the optimal clinical settings for this type of testing are limited. We performed a retrospective study at Mayo Clinic, Rochester, Minnesota, USA, with typically sterile specimens from children that underwent 16S rRNA PCR testing during September 2020–December 2023. Of 162 tests performed on 124 patients, 20% were positive; 58% of positive samples were from culture-negative specimens. Fluid specimens were >3 times as likely to test positive as tissue specimens (odds ratio 3.07 [95% CI 1.32–7.11]; p = 0.007), and pleural fluid demonstrated the highest positivity rate (50%). Of 33 positive results, 4 (12%) specimens qualified for reporting to the state health department for communicable diseases. Those single-laboratory findings demonstrate that the highest positivity rate of 16S rRNA PCR and sequencing is pleural fluid, although many specimen types tested positive.