During routine clinical practice, infectious disease physicians encounter patients with difficult-to-diagnose clinical syndromes and may order advanced molecular testing to detect pathogens. These tests may identify potential infectious causes for illness and allow clinicians to adapt treatments or stop unnecessary antimicrobials. Cases of pathogen-agnostic disease testing also provide an important window into known, emerging, and reemerging pathogens and may be leveraged as part of national sentinel surveillance. A survey of Emerging Infections Network members, a group of infectious disease providers in North America, was conducted in May 2023. The objective of the survey was to gain insight into how and when infectious disease physicians use advanced molecular testing for patients with difficult-to-diagnose infectious diseases, as well as to explore the usefulness of advanced molecular testing and barriers to use. Overall, 643 providers answered at least some of the survey questions; 478 (74%) of those who completed the survey had ordered advanced molecular testing in the last two years, and formed the basis for this study. Respondents indicated that they most often ordered broad-range 16S rRNA gene sequencing, followed by metagenomic next-generation sequencing and whole genome sequencing; and commented that in clinical practice, some, but not all tests were useful. Many physicians also noted several barriers to use, including a lack of national guidelines and cost, while others commented that whole genome sequencing had potential for use in outbreak surveillance. Improving frontline physician access, availability, affordability, and developing clear national guidelines for interpretation and use of advanced molecular testing could potentially support clinical practice and public health surveillance.

Clade I monkeypox virus (MPXV), which can cause severe illness in more people than clade II MPXVs, is endemic in the Democratic Republic of the Congo (DRC), but the country has experienced an increase in suspected cases during 2023-2024. In light of the 2022 global outbreak of clade II mpox, the increase in suspected clade I cases in DRC raises concerns that the virus could spread to other countries and underscores the importance of coordinated, urgent global action to support DRC's efforts to contain the virus. To date, no cases of clade I mpox have been detected outside of countries in Central Africa where the virus is endemic. CDC and other partners are working to support DRC's response. In addition, CDC is enhancing U.S. preparedness by raising awareness, strengthening surveillance, expanding diagnostic testing capacity for clade I MPXV, ensuring appropriate specimen handling and waste management, emphasizing the importance of appropriate medical treatment, and communicating guidance on the recommended contact tracing, containment, behavior modification, and vaccination strategies.

To better identify emerging or reemerging pathogens in patients with difficult-to-diagnose infections, it is important to improve access to advanced molecular testing methods. This is particularly relevant for cases where conventional microbiologic testing has been unable to detect the pathogen and the patient's specimens test negative. To assess the availability and utility of such testing for human clinical specimens, a literature review of published biomedical literature was conducted. From a corpus of more than 4,000 articles, a set of 34 reports was reviewed in detail for data on where the testing was being performed, types of clinical specimens tested, pathogen agnostic techniques and methods used, and results in terms of potential pathogens identified. This review assessed the frequency of advanced molecular testing, such as metagenomic next generation sequencing that has been applied to clinical specimens for supporting clinicians in caring for difficult-to-diagnose patients. Specimen types tested were from cerebrospinal fluid, respiratory secretions, and other body tissues and fluids. Publications included case reports and series, and there were several that involved clinical trials, surveillance studies, research programs, or outbreak situations. Testing identified both known human pathogens (sometimes in new sites) and previously unknown human pathogens. During this review, there were no apparent coordinated efforts identified to develop regional or national reports on emerging or reemerging pathogens. Therefore, development of a coordinated sentinel surveillance system that applies advanced molecular methods to clinical specimens which are negative by conventional microbiological diagnostic testing would provide a foundation for systematic characterization of emerging and underdiagnosed pathogens and contribute to national biodefense strategy goals.

The surveillance and identification of emerging, reemerging, and unknown infectious disease pathogens is essential to national public health preparedness and relies on fluidity, coordination, and interconnectivity between public and private pathogen surveillance systems and networks. Developing a national sentinel surveillance network with existing resources and infrastructure could increase efficiency, accelerate the identification of emerging public health threats, and support coordinated intervention strategies that reduce morbidity and mortality. However, implementing and sustaining programs to detect emerging and reemerging pathogens in humans using advanced molecular methods, such as metagenomic sequencing, requires making large investments in testing equipment and developing networks of clinicians, laboratory scientists, and bioinformaticians. In this study, we sought to gain an understanding of how federal government agencies currently support such pathogen agnostic testing of human specimens in the United States. We conducted a landscape analysis of federal agency websites for publicly accessible information on the availability and type of pathogen agnostic testing and details on flow of clinical specimens and data. The website analysis was supplemented by an expert review of results with representatives from the federal agencies. Operating divisions within the US Department of Health and Human Services and the US Department of Veterans Affairs have developed and sustained extensive clinical and research networks to obtain patient specimens and perform metagenomic sequencing. Metagenomic facilities supported by US agencies were not equally geographically distributed across the United States. Although many entities have work dedicated to metagenomics and/or support emerging infectious disease surveillance specimen collection, there was minimal formal collaboration across agencies.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.One sentence summary Protein complementation enables mix-and-read SARS-CoV-2 serology that rivals sensitivity and specificity of ELISA but excels in throughput and quantitation.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis research was funded by the Centers for Disease Control and Prevention.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Residual specimen materials were used for diagnostics development under a protocol that was reviewed and approved by the CDC Institutional Review Board (See 45 C.F.R. part 46; 21 C.F.R. part 56)All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesNo external data links

Self-tests* to detect current infection with SARS-CoV-2, the virus that causes COVID-19, are valuable tools that guide individual decision-making and risk reduction() (1-3). Increased self-test use (4) has likely contributed to underascertainment of COVID-19 cases (5-7), because unlike the requirements to report results of laboratory-based and health care provider-administered point-of-care COVID-19 tests,() public health authorities do not require reporting of self-test results. However, self-test instructions include a recommendation that users report results to their health care provider so that they can receive additional testing and treatment if clinically indicated.() In addition, multiple manufacturers of COVID-19 self-tests have developed websites or companion mobile applications for users to voluntarily report self-test result data. Federal agencies use the data reported to manufacturers, in combination with manufacturing supply chain information, to better understand self-test availability and use. This report summarizes data voluntarily reported by users of 10.7 million self-tests from four manufacturers during October 31, 2021-June 11, 2022, and compares these self-test data with data received by CDC for 361.9 million laboratory-based and point-of-care tests performed during the same period. Overall trends in reporting volume and percentage of positive results, as well as completeness of reporting demographic variables, were similar across test types. However, the limited amount and quality of data reported from self-tests currently reduces their capacity to augment existing surveillance. Self-tests provide important risk-reduction information to users, and continued development of infrastructure and methods to collect and analyze data from self-tests could improve their use for surveillance during public health emergencies.

Serosurveillance can provide estimates of population-level exposure to infectious pathogens and has been used extensively during the COVID-19 pandemic. Simultaneous, serological testing for multiple pathogens can be done using bead-based immunoassays to add value to disease-specific serosurveys. We conducted a validation of four SARS-CoV-2 antigens-full-length spike protein, two receptor binding domain proteins, and the nucleocapsid protein-on our existing multiplex bead assay (MBA) for enteric diseases, malaria, and vaccine preventable diseases. After determining the optimal conditions for coupling the antigens to microsphere beads, the sensitivity and specificity of the assay were determined on two instruments (Luminex-200 and MAGPIX) when testing singly (monoplex) versus combined (multiplex). Sensitivity was assessed using plasma from 87 real-time reverse transcription polymerase chain reaction (rRT-PCR) positive persons collected in March-May of 2020 and ranged from 94.3% to 96.6% for the different testing conditions. Specificity was assessed using 98 plasma specimens collected prior to December 2019 and plasma from 19 rRT-PCR negative persons and ranged from 97.4% to 100%. The positive percent agreement was 93.8% to 97.9% using 48 specimens collected > 21 days post-symptom onset, while the negative percent agreement was ≥ 99% for all antigens. Test performance was similar using monoplex or multiplex testing. Integrating SARS-CoV-2 serology with other diseases of public health interest could add significant value to public health programs that have suffered severe programmatic setbacks during the COVID-19 pandemic.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

The public health community has recognized that it cannot handle responses to all possible public health emergencies on its own. The public health sector has deep scientific expertise and excels at initial identification, complex characterization, and test development. The private sector has many resources and capabilities that can complement and augment the public health response. This is especially true in the clinical laboratory sector. Many commercial laboratories are designed for high-volume, high-throughput diagnostic testing in a way that public health laboratories are not. Significant steps have been taken since 2017 to improve the communication and coordination between public health and the private clinical laboratory community, especially during a response to a public health emergency. This paper describes the strong foundation that has been built for an improved clinical and public health laboratory response to the next public health emergency.

Sample collection procedures and primary receptacle (sample container and bag) decontamination methods should prevent contaminant transfer between contaminated and non-contaminated surfaces and areas during bio-incident operations. Cross-contamination of personnel, equipment, or sample containers may result in the exfiltration of biological agent from the exclusion (hot) zone and have unintended negative consequences on response resources, activities and outcomes. The current study was designed to 1) evaluate currently recommended sample collection and packaging procedures to identify procedural steps that may increase the likelihood of spore exfiltration or contaminant transfer, 2) evaluate the efficacy of currently recommended primary receptacle decontamination procedures, and 3) evaluate the efficacy of outer packaging decontamination methods. Wet- and dry-deposited fluorescent tracer powder was used in contaminant transfer tests to qualitatively evaluate the currently-recommended sample collection procedures. Bacillus atrophaeus spores, a surrogate for Bacillus anthracis, were used to evaluate the efficacy of spray- and wipe-based decontamination procedures. Both decontamination procedures were quantitatively evaluated on three types of sample packaging materials (corrugated fiberboard, polystyrene foam, and polyethylene plastic), and two contamination mechanisms (wet or dry inoculums). Contaminant transfer results suggested that size-appropriate gloves should be worn by personnel, templates should not be taped to or removed from surfaces, and primary receptacles should be selected carefully. The decontamination tests indicated that wipe-based decontamination procedures may be more effective than spray-based procedures; efficacy was not influenced by material type but was affected by the inoculation method. Incomplete surface decontamination was observed in all tests with dry inoculums. This study provides a foundation for optimizing current B. anthracis response procedures to minimize contaminant exfiltration.