A Comprehensive Guide to Quality Assessment and Data Submission for Genomic Surveillance of Enteric Pathogens.

This document outlines the steps necessary to assemble and submit the standard data package required for contributing to the global genomic surveillance of enteric pathogens. Although targeted to GenomeTrakr laboratories and collaborators, these protocols are broadly applicable for enteric pathogens collected for different purposes. There are five protocols included in this chapter: (1) quality control (QC) assessment for the genome sequence data, (2) validation for the contextual data, (3) data submission for the standard pathogen package or Pathogen Data Object Model (DOM) to the public repository, (4) viewing and querying data at NCBI, and (5) data curation for maintaining relevance of public data. The data are available through one of the International Nucleotide Sequence Database Consortium (INSDC) members, with the National Center for Biotechnology Information (NCBI) being the primary focus of this document. NCBI Pathogen Detection is a custom dashboard at NCBI that provides easy access to pathogen data plus results for a standard suite of automated cluster and genotyping analyses important for informing public health and regulatory decision-making.

An assessment of excess mortality during the COVID-19 pandemic, a retrospective post-mortem surveillance in 12 districts - Zambia, 2020-2022.

BACKGROUND: The number of COVID-19 deaths reported in Zambia (N = 4069) is most likely an underestimate due to limited testing, incomplete death registration and inability to account for indirect deaths due to socioeconomic disruption during the pandemic. We sought to assess excess mortality during the COVID-19 pandemic in Zambia. METHODS: We conducted a retrospective analysis of monthly-death-counts (2017-2022) and individual-daily-deaths (2020-2022) of all reported health facility and community deaths at district referral health facility mortuaries in 12 districts in Zambia. We defined COVID-19 wave periods based on a sustained nationally reported SARS-CoV-2 test positivity of greater than 5%. Excess mortality was calculated as the difference between observed monthly death counts during the pandemic (2020-2022) and the median monthly death counts from the pre-pandemic period (2017-2019), which served as the expected number of deaths. This calculation was conducted using a Microsoft Excel-based tool. We compared median daily death counts, median age at death, and the proportion of deaths by place of death (health facility vs. community) by wave period using the Mann-Whitney-U test and chi-square test respectively in R. RESULTS: A total of 112,768 deaths were reported in the 12 districts between 2020 and 2022, of which 17,111 (15.2%) were excess. Wave periods had higher median daily death counts than non-wave periods (median [IQR], 107 [95-126] versus 96 [85-107], p < 0.001). The median age at death during wave periods was older than non-wave periods (44.0 [25.0-67.0] versus 41.0 [22.0-63.0] years, p < 0.001). Approximately half of all reported deaths occurred in the community, with an even greater proportion during wave periods (50.6% versus 53.1%, p < 0.001), respectively. CONCLUSION: There was excess mortality during the COVID-19 pandemic in Zambia, with more deaths occurring within the community during wave periods. This analysis suggests more COVID-19 deaths likely occurred in Zambia than suggested by officially reported numbers. Mortality surveillance can provide important information to monitor population health and inform public health programming during pandemics.

Public Mass Shootings: Counterfactual Trend Analysis of the Federal Assault Weapons Ban.

Background: Assault weapon and large-capacity magazine bans are potential tools for policy makers to prevent public mass shootings. However, the efficacy of these bans is a continual source of debate. In an earlier study, we estimated the impact of the Federal Assault Weapons Ban (FAWB) on the number of public mass shooting events in the United States. This study provides an updated assessment with 3 additional years of firearm surveillance data to characterize the longer-term effects. Objective: This study aims to estimate the impact of the FAWB on trends in public mass shootings from 1966 to 2022. Methods: We used linear regression to estimate the impact of the FAWB on the 4-year simple moving average of annual public mass shootings, defined by events with 4 or more deaths in 24 hours, not including the perpetrator. The study period spans 1966 to 2022. The model includes indicator variables for both the FAWB period (1995-2004) and the period after its removal (2005-2022). These indicators were interacted with a linear time trend. Estimates were controlled for the national homicide rate. After estimation, the model provided counterfactual estimates of public mass shootings if the FAWB was never imposed and if the FAWB remained in place. Results: The overall upward trajectory in the number of public mass shootings substantially fell while the FAWB was in place. These trends are specific to events in which the perpetrator used an assault weapon or large-capacity magazine. Point estimates suggest the FAWB prevented up to 5 public mass shootings while the ban was active. A continuation of the FAWB and large-capacity magazine ban would have prevented up to 38 public mass shootings, but the CIs become wider as time moves further away from the period of the FAWB. Conclusions: The FAWB, which included a ban on large-capacity magazines, was associated with fewer public mass shooting events, fatalities, and nonfatal gun injuries. Gun control legislation is an important public health tool in the prevention of public mass shootings.

Detection of circulating tumor DNA by tumor-informed whole-genome sequencing enables prediction of recurrence in stage III colorectal cancer patients.

INTRODUCTION: Circulating tumor (ctDNA) can be used to detect residual disease after cancer treatment. Detecting low-level ctDNA is challenging, due to the limited number of recoverable ctDNA fragments at any target loci. In response, we applied tumor-informed whole-genome sequencing (WGS), leveraging thousands of mutations for ctDNA detection. METHODS: Performance was evaluated in serial plasma samples (n = 1283) from 144 stage III colorectal cancer patients. Tumor/normal WGS was used to establish a patient-specific mutational fingerprint, which was searched for in 20x WGS plasma profiles. For reproducibility, paired aliquots of 172 plasma samples were analyzed in two independent laboratories. De novo variant calling was performed for serial plasma samples with a ctDNA level > 10 % (n = 17) to explore genomic evolution. RESULTS: WGS-based ctDNA detection was prognostic of recurrence: post-operation (Hazard ratio [HR] 6.75, 95 %CI 3.18-14.3, p < 0.001), post-adjuvant chemotherapy (HR 28.9, 95 %CI 10.1-82.8; p < 0.001), and during surveillance (HR 22.8, 95 %CI 13.7-37.9, p < 0.0001). The 3-year cumulative incidence of ctDNA detection in recurrence patients was 95 %. ctDNA was detected a median of 8.7 months before radiological recurrence. The independently analyzed plasma aliquots showed excellent agreement (Cohens Kappa=0.9, r = 0.99). Genomic characterization of serial plasma revealed significant evolution in mutations and copy number alterations, and the timing of mutational processes, such as 5-fluorouracil-induced mutations. CONCLUSION: Our study supports the use of WGS for sensitive ctDNA detection and demonstrates that post-treatment ctDNA detection is highly prognostic of recurrence. Furthermore, plasma WGS can identify genomic differences distinguishing the primary tumor and relapsing metastasis, and monitor treatment-induced genomic changes.

Age- and Sex-Specific Differences in Lyme Disease Health-Related Behaviors, Ontario, Canada, 2015-2022.

We investigated differences in risk factors and preventive behaviors by age and sex among persons with reported Lyme disease in Ontario, Canada, during 2015-2022. Incidence rates peaked among children 5-9 and adults 50-79 years of age. Median age was higher for female than male case-patients (54 vs. 51 years). Male case-patients reported more activity in wooded and tall grass areas than did female case-patients; fewer male case-patients reported sharing living space with outdoor-exposed companion animals. As age increased, more case-patients reported activity in blacklegged tick habitats, exposure to ticks, and wearing adequate clothing, but fewer reported sharing living space with outdoor-exposed companion animals. Adoption of preventive behaviors was relatively low and did not differ by sex. Male case-patients, children 5-9 years of age and their parents or caregivers, and adults >59 years of age represent populations that would benefit from tailored public health messaging on Lyme disease prevention.

Early Introductions of Candida auris Detected by Wastewater Surveillance, Utah, USA, 2022-2023.

Candida auris is considered a nosocomial pathogen of high concern and is currently spreading across the United States. Infection control measures for C. auris focus mainly on healthcare facilities, yet transmission levels may already be significant in the community before outbreaks are detected in healthcare settings. Wastewater-based epidemiology (culture, quantitative PCR, and whole-genome sequencing) can potentially gauge pathogen transmission in the general population and lead to early detection of C. auris before it is detected in clinical cases. To learn more about the sensitivity and limitations of wastewater-based surveillance, we used wastewater-based methods to detect C. auris in a southern Utah jurisdiction with no known clinical cases before and after the documented transfer of colonized patients from bordering Nevada. Our study illustrates the potential of wastewater-based surveillance for being sufficiently sensitive to detect C. auris transmission during the early stages of introduction into a community.

Infectious Disease Physicians' Knowledge and Practices Regarding Wastewater Surveillance, United States, 2024.

A survey of US infectious disease physicians indicated that few regularly reviewed wastewater surveillance (WWS) data but many reported examples of how WWS has affected or could affect their clinical practice. WWS data can be useful for physicians, but increased communication between public health professionals and physicians regarding WWS could improve its utility.

Correlation between Viral Wastewater Concentration and Respiratory Tests, Oregon, USA.

We evaluated the association between wastewater concentration and weekly percent positivity of patient testing for SARS-CoV-2, influenza, and respiratory syncytial virus in Oregon, USA. We found strong, positive correlations for SARS-CoV-2 (ρ = 0.84, p<0.001), influenza (ρ = 0.73, p<0.001) and respiratory syncytial virus (ρ = 0.69, p<0.001).

Evidence of Lineage 1 and 3 West Nile Virus in Person with Neuroinvasive Disease, Nebraska, USA, 2023.

West Nile virus (WNV) is the most common cause of human arboviral disease in the contiguous United States, where only lineage 1 (L1) WNV had been found. In 2023, an immunocompetent patient was hospitalized in Nebraska with West Nile neuroinvasive disease and multisystem organ failure. Testing at the Centers for Disease Control and Prevention indicated an unusually high viral load and acute antibody response. Upon sequencing of serum and cerebrospinal fluid, we detected lineage 3 (L3) and L1 WNV genomes. L3 WNV had previously only been found in Central Europe in mosquitoes. The identification of L3 WNV in the United States and the observed clinical and laboratory features raise questions about the potential effect of L3 WNV on the transmission dynamics and pathogenicity of WNV infections. Determining the distribution and prevalence of L3 WNV in the United States and any public health and clinical implications is critical.

Bluetongue Virus in the Iberian Lynx (Lynx pardinus), 2010-2022.

Clinical infection and death caused by bluetongue virus infection has been reported in the Eurasian lynx. Bluetongue virus surveillance in the Iberian lynx revealed widespread and repeated exposure to serotypes 1 and 4 in wild and captive populations of this species. This exposure is possibly from a spillover event from sympatric ruminants.

Severe acute respiratory syndrome coronavirus 2-reactive salivary antibody detection in South Carolina emergency healthcare workers, September 2019-March 2020.

On 19 January 2020, the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was identified in the United States, with the first cases in South Carolina confirmed on 06 March 2020. Due to initial limited testing capabilities and potential for asymptomatic transmission, it is possible that SARS-CoV-2 may have been present earlier than previously thought, while the immune status of at-risk populations was unknown. Saliva from 55 South Carolina emergency healthcare workers (EHCWs) was collected from September 2019 to March 2020, pre- and post-healthcare shifts, and stored frozen. To determine the presence of SARS-CoV-2-reactive antibodies, saliva-acquired post-shift was analysed by enzyme-linked immunosorbent assay (ELISA) with a repeat of positive or inconclusive results and follow-up testing of pre-shift samples. Two participants were positive for SARS-CoV-2 N/S1-reactive IgG, confirmed by follow-up testing, with S1 receptor binding domain (RBD)-specific IgG present in one individual. Positive samples were collected from medical students working in emergency medical services (EMSs) in October or November 2019. The presence of detectable anti-SARS-CoV-2 antibodies in 2019 suggests that immune responses to the virus existed in South Carolina, and the United States, in a small percentage of EHCWs prior to the earliest documented coronavirus disease 2019 (COVID-19) cases. These findings suggest the feasibility of saliva as a noninvasive tool for surveillance of emerging outbreaks, and EHCWs represent a high-risk population that should be the focus of infectious disease surveillance.

Tumor Deposits as an Adverse Prognostic Indicator in Stage III Colon Cancer: A Multicenter Database Study.

Objective: We explored the oncological impact of tumor deposits (TDs) on colon cancer and proposed optimal modifications to the current staging system. Background: In the existing American Joint Committee on Cancer colon cancer staging system, TDs are incorporated into the N category as N1c. When lymph node metastases (LNMs) are present, their number is considered to determine nodal stages, such as N1a/b or N2a/b, regardless of TDs. Methods: 4212 patients with primary colon cancer who underwent surgical resection in the Seoul Colorectal Group (2010-2020) and 93,057 patients from the Surveillance, Epidemiology, and End Results*Stat database (2000-2017) were included in this study. Patients were classified according to the number of metastatic lymph nodes (LNs) (0/1-3/≥4) and the presence of TDs. Results: TDs were significantly associated with left colon cancer, a higher T category, and vascular/perineural invasion. Patients with TDs had higher recurrence rates (23.1 vs 7.5%, P < 0.001). The TD-positive patients had notably worse overall survival (OS) and recurrence-free survival rates. The survival outcomes of TD-positive patients without LNM were inferior to those of TD-negative patients with LN1-3 (5-year OS: 78.9 vs 87.8%, P = 0.04). The survival outcomes of TD-positive patients with LN1-3 were similar to those of TD-negative patients with LN ≥4 (5-year OS: 87.0 vs 77.1%, P = 0.11). Survival outcomes obtained using the Surveillance, Epidemiology, and End Results *Stat database yielded consistent results. Conclusions: TDs were associated with poor prognostic factors and had a significant impact on survival outcomes. The incorporation of tumor deposits into nodal classifications beyond the current N1c criteria may improve the staging system and more accurately reflect the recurrence and survival rates among patients with colon cancer. TD-positive in N1a or N1b could be categorized as N2.

Choosing Your Endoscopist: A Retrospective Single-Centre Cohort Study.

Background Colorectal cancer is one of the most common internal malignancies affecting Australians, and colonoscopy is widely accepted as a part of comprehensive large bowel assessment. Different specialties perform colonoscopies, most commonly general surgeons and gastroenterologists. Analysing performance outcomes against benchmarks allows insight into inter-specialty differences and enables the improvement of overall service provision and quality. Methods We performed a retrospective single-centre cohort study on 2086 patients undergoing colonoscopies by seven surgeons (S) and nine gastroenterologists (G) between July 2021 and June 2023. Primary outcomes were comparative caecal intubation rates (CIR), photo documentation rates (PDR), documented withdrawal rates (DWR), withdrawal times (WT), and adenoma detection rates (ADR). Secondary outcomes characterised adenoma frequency, optimal WT, and indications for colonoscopies. Results We found significant differences in CIR (S: 94.9%, 990/1043; G: 99%, 1033/1043, P<0.01), PDR (S: 95.9%, 949/990; G: 99.1%, 1024/1033, P<0.01), DWR (S: 17.4%, 181/1043; G: 87.3%, 911/1043, P<0.01), WT >6 minutes (S: 82.3%, 149/181; G: 97.8%, 891/911, P<0.01), and ADR (S: 37.9%, 193/509; G: 59.7%, 421/705, P<0.01). Subgroup analysis revealed adenoma frequency peaked at 50-70 years old and optimal WT was ≥9 minutes. We demonstrated surgeons mainly perform colonoscopies for diverticulitis surveillance, abnormal imaging, post-cancer resections, and rectal bleeding, but gastroenterologists predominantly investigate bowel symptoms, polyp surveillance, positive faecal occult blood test, and anaemia. Conclusion Despite both specialties surpassing national standards in CIR and ADR, there were significant differences in performance indicators. We believe ADR differences could be explained by different indications specialties perform colonoscopies for. Increasing WT ≥9 minutes could improve ADR, and education on the usage of withdrawal timer on endoscopes will improve DWR.

The rising concern of Oropouche fever: a call for enhanced surveillance and research in emerging arboviral threats.

AIM OF STUDY: The study aims to assess the growing public health threat posed by Oropouche virus (OROV), focusing on its epidemiology, transmission patterns, and the challenges in diagnosis and control. By analyzing the recent spread of OROV to new regions, including Cuba and Colombia, the study seeks to highlight the need for improved surveillance, enhanced diagnostic capabilities, and research into potential treatments and vaccines. Additionally, the study investigates the clinical similarities between Oropouche fever and other arboviruses, which often lead to diagnostic difficulties and mismanagement in affected regions. RESULTS: The virus has caused over 500,000 cases in Brazil alone, with recent outbreaks reporting fatalities, suspected vertical transmission, and potential associations with microcephaly in newborns. Underreporting and limited surveillance have likely led to the underestimation of the true burden of Oropouche fever. Current diagnostic methods, such as serology and RT-PCR, are often inaccessible in low-resource settings, further complicating efforts to control the spread of the virus. The study highlights the importance of improving diagnostic capacity, enhancing surveillance, and conducting further research into vector control, antiviral treatments, and vaccine development. CONCLUSION: This study emphasizes the urgent need for coordinated international efforts to address the rising threat of Oropouche virus. Considering its rapid spread and potential for global transmission, comprehensive public health measures are necessary to protect vulnerable populations and mitigate the impact of this emerging disease. Enhanced surveillance and the development of accessible diagnostics, vaccines, and treatment options are critical to containing OROV and preventing further outbreaks.

Early detection and prediction of non-fatal drug-related incidents and fatal overdose outbreaks using the Farrington algorithm.

AIMS: The aim of this study was to assess the validity of undertaking time-series analyses on both fatal and non-fatal drug overdose outcomes for the surveillance of emerging drug threats, and to determine the validity of analyzing non-fatal indicators to support the early detection of fatal overdose outbreaks. DESIGN, SETTING AND PARTICIPANTS: Time-series analyses using county-level data containing fatal overdoses and non-fatal overdose counts were collected at monthly intervals between 2015 and 2021 in California and Florida, USA. To analyze these data, we used the Farrington algorithm (FA), a method used to detect aberrations in time-series data such that an abnormal increase in counts relative to previous observations would result in an alert. The FA's performance was compared with a bench-mark approach, using the standard deviation as an aberration detection threshold. We evaluated whether monthly alerts in non-fatal overdose can aid in identifying fatal drug overdose outbreaks, defined as a statistically significant increase in the 6-month overdose death rate. We also conducted analyses across regions, i.e. clusters of counties. MEASUREMENTS: Measurements were taken during emergency department and emergency medical service visits. FINDINGS: Both methods yielded a similar proportion of alerts across scenarios for non-fatal overdoses, while the bench-mark method yielded more alerts for fatal overdoses. For both methods, the correlations between surveillance evaluations were relatively poor in the detection of aberrations (typically < 35%) but were high between evaluations yielding no alerts (typically > 75%). For ongoing fatal overdose outbreaks, a strategy based on the detection of alerts at the county level from either method yielded a sensitivity of 66% for both California and Florida. At the regional level, the equivalent analyses had sensitivities of 81% for California and 77% for Florida. CONCLUSION: Aberration detection methods can support the early detection of fatal drug overdose outbreaks, particularly when methodologies are applied in combination rather than individual methods separately.

The French registry of sudden unexpected death in infancy (SUDI): a 7-year review of available data.

The French "OMIN registry" was established in 2015 to collect nationwide standardised data concerning biological, clinical, environmental and social characteristics of sudden unexpected death in infancy (SUDI) and unexpected death in children aged 1-2 years. A biobank has existed since July 2020 to store biological samples for each case. This article aimed to detail (1) a brief history and the objectives of the registry; (2) a description of the methodology used; (3) the first results of the registry, i.e. the main characteristics of the cases included so far; (4) the process for accessing the data for research projects; and (5) issues regarding weakness and improvement and perspectives offered by the registry. On 31 May 2024, 1975 cases were included in the OMIN registry; on 31 December 2022, 4606 biological samples from 176 cases were collected. For each deceased child, different types of data are registered on an electronic case report form: socio-demographic data, personal and familial medical background, environment and feeding data, clinical data, and biological and imaging results. A strict and continuous quality control process is used to ensure the reliability of the data, in parallel with specific actions to improve the exhaustiveness of the registry. The OMIN registry database is one of the largest and the most complete databases on SUDI, especially in Europe, and the first in the world to associate a standardised biological sample collection with it. Perspectives of research provided by our registry are numerous and could be supported by national and international scientific collaborations. CONCLUSION:  This article details the objectives and methods of the French registry of SUDI. It provides initial results relating to the population included in the register and the procedure for accessing the data. WHAT IS KNOWN: • In Western Europe, France is one of the countries with the highest SUDI rate, making it the first cause of death of infants between 28 and 364 days. • The development of epidemiological tools on a national and international scale is essential to advance research into the determinants and risk factors of unexpected death in children under 2 years of age. WHAT IS NEW: • The OMIN registry was created in France in 2015 to collect nationwide standardised social, environmental, clinical, and paraclinical data for cases of unexpected death in children aged 0 to 2 years. • To date, the OMIN registry has included 680 data from almost 2000 children unexpectedly deceased, completed by a biocollection since 2020. • Data from the OMIN registry, unique in its field, are freely available for scientific research teams, after acceptation by the scientific committee of the registry.

Monitoring the sleep health of adults: a scoping review of routine national surveillance systems.

Study Objectives: The aims of this review were to identify existing national surveillance systems monitoring one or more domains of sleep health in adults, and to describe the specific sleep health indicators used. Methods: We systematically searched the gray and peer-reviewed literature for routinely conducted cross-sectional and longitudinal nationally representative health surveys that included the assessment of at least one domain of sleep health. The methodology involved: (1) targeted searches of the websites of national and international health agencies and statistics departments for 199 countries, (2) country-specific customized internet searches, and (3) country-specific electronic database searches of PubMed. Results: A total of 19 762 records were identified from both the gray and peer-reviewed literature. Sleep health surveillance at the national level was conducted by 51 countries (25.6%) across 69 national health surveys. Sleep quality (96.1% of countries that surveilled sleep) was the most frequently assessed followed by sleep duration (27.5%), sleep medication use (25.5%), sleep disorders (17.6%), daytime alertness (15.7%), sleep satisfaction (15.7%), and sleep timing (7.8%). Additionally, 34.8% of the surveys utilized multiple sleep health indicators. Conclusions: This study identified three significant gaps in the coverage of sleep health within national surveillance systems. Limited population sleep data in low- and middle-income countries, inconsistent use of sleep-related items in surveys and questionnaires, and substantial variability in the definitions of sleep health indicators. Advocacy for the inclusion of sleep health within national surveillance systems may be warranted given the important role sleep plays in public health.

iNaturalist community observations provide valuable data on human-mosquito encounters.

Mosquitoes (Diptera: Culicidae) and the pathogens they transmit represent a threat to human and animal health. Low-cost and effective surveillance methods are necessary to enable sustainable monitoring of mosquito distributions, diversity, and human interactions. This study examined the use of iNaturalist, an online, community-populated biodiversity recording database, for passive mosquito surveillance in the United Kingdom (UK) and Ireland, countries under threat from the introduction of invasive mosquitoes and emerging mosquito-borne diseases. The Mozzie Monitors UK & Ireland iNaturalist project was established to collate mosquito observations in these countries. Data were compared with existing long-term mosquito UK datasets to assess representativeness of seasonal and distribution trends in citizen scientist-recorded observations. The project collected 738 observations with the majority recorded 2020-2022. Records were primarily associated with urban areas, with the most common species Culex pipiens and Culiseta annulata significantly more likely to be observed in urban areas than other species. Analysis of images uploaded to the iNaturalist project also provided insights into human-biting behavior. Our analyses indicate that iNaturalist provides species composition, seasonal occurrence, and distribution figures consistent with existing datasets and is therefore a useful surveillance tool for recording information on human interactions with mosquitoes and monitoring species of concern.

Advancing Public Health Surveillance in Child Care Centers: Stakeholder-Informed Redesign and User Satisfaction Evaluation of the MCRISP Network.

Unlabelled: Leveraging user feedback, we redesigned a novel disease monitoring utility to allow for bidirectional data flow and in this letter offer insights into that process as well as lessons learned.

Assessment of the Effective Sensitivity of SARS-CoV-2 Sample Pooling Based on a Large-Scale Screening Experience: Retrospective Analysis.

BACKGROUND: The development of new large-scale saliva pooling detection strategies can significantly enhance testing capacity and frequency for asymptomatic individuals, which is crucial for containing SARS-CoV-2. OBJECTIVE: This study aims to implement and scale-up a SARS-CoV-2 screening method using pooled saliva samples to control the virus in critical areas and assess its effectiveness in detecting asymptomatic infections. METHODS: Between August 2020 and February 2022, our laboratory received a total of 928,357 samples. Participants collected at least 1 mL of saliva using a self-sampling kit and registered their samples via a smartphone app. All samples were directly processed using AutoMate 2550 for preanalytical steps and then transferred to Microlab STAR, managed with the HAMILTON Pooling software for pooling. The standard pool preset size was 20 samples but was adjusted to 5 when the prevalence exceeded 2% in any group. Real-time polymerase chain reaction (RT-PCR) was conducted using the Allplex SARS-CoV-2 Assay until July 2021, followed by the Allplex SARS-CoV-2 FluA/FluB/RSV assay for the remainder of the study period. RESULTS: Of the 928,357 samples received, 887,926 (95.64%) were fully processed into 56,126 pools. Of these pools, 4863 tested positive, detecting 5720 asymptomatic infections. This allowed for a comprehensive analysis of pooling's impact on RT-PCR sensitivity and false-negative rate (FNR), including data on positive samples per pool (PPP). We defined Ctref as the minimum cycle threshold (Ct) of each data set from a sample or pool and compared these Ctref results from pooled samples with those of the individual tests (ΔCtP). We then examined their deviation from the expected offset due to dilution [ΔΔCtP = ΔCtP - log2]. In this work, the ΔCtP and ΔΔCtP were 2.23 versus 3.33 and -0.89 versus 0.23, respectively, comparing global results with results for pools with 1 positive sample per pool. Therefore, depending on the number of genes used in the test and the size of the pool, we can evaluate the FNR and effective sensitivity (1 - FNR) of the test configuration. In our scenario, with a maximum of 20 samples per pool and 3 target genes, statistical observations indicated an effective sensitivity exceeding 99%. From an economic perspective, the focus is on pooling efficiency, measured by the effective number of persons that can be tested with 1 test, referred to as persons per test (PPT). In this study, the global PPT was 8.66, reflecting savings of over 20 million euros (US $22 million) based on our reagent prices. CONCLUSIONS: Our results demonstrate that, as expected, pooling reduces the sensitivity of RT-PCR. However, with the appropriate pool size and the use of multiple target genes, effective sensitivity can remain above 99%. Saliva pooling may be a valuable tool for screening and surveillance in asymptomatic individuals and can aid in controlling SARS-CoV-2 transmission. Further studies are needed to assess the effectiveness of these strategies for SARS-CoV-2 and their application to other microorganisms or biomarkers detected by PCR.