Optimizing congenital cytomegalovirus detection by pool testing in saliva by a rapid molecular test.

Universal congenital cytomegalovirus (cCMV) screening in saliva is increasingly recommended. The aim of our study was to correlate the performance of a point-of-care rapid molecular test with CMV real time PCR (CMV RT-PCR) detection, using saliva pool-testing in newborns under a universal screening strategy. Saliva swabs were prospectively collected from newborns < 21 days old and tested by Alethia-LAMP-CMV assay in pools of 5 samples. In positive pools, subjects were tested individually and by saliva and urine CMV RT-PCR. A subset of negative pools were studied with both techniques and viral loads in whole blood were determined in positive patients. From 1,642 newborns included in 328 pools, 8 were confirmed by urine CMV RT-PCR, (cCMV prevalence 0,49%). The PPA and NNA of the pooled saliva Alethia-LAMP-CMV testing were 87,5% and 99,8% with a negative and positive predictive value of 99,9% and 77,7%, respectively. Two false positives were detected (0,12%). A subset of 17 negative pools (85 samples), studied by saliva CMV RT-PCR, showed 100% concordance.  Conclusion: CMV pool-testing using a rapid molecular test in saliva proved feasible when compared to PCR gold standards. This strategy could improve cost-effectiveness for cCMV universal neonatal screening, based on the low prevalence of the infection and could be a more affordable approach in less developed regions with reduced detection capacity. What is Known: • cCMV is the most frequent congenital infection and a leading nongenetic cause of sensorineural hearing loss and brain disease. • Universal screening could allow early detection of congenitally infected infants, improving clinical outcome. • Saliva PCR is the preferred and non-invasive test for newborn cCMV screening. What is New: • The feasibility of a universal cCMV screening by pool-testing in saliva using a rapid test in pools of 5 samples. • PPA and NPA were 87,5 and 99,8% compared to CMV PCR in urine. • This strategy could be relevant specially in LMIC where detection capacity is reduced and could improve cost-effectiveness. • cCMV prevalence in our center was 0,49%.

Pool testing with dilution effects and heterogeneous priors.

The Dorfman pooled testing scheme is a process in which individual specimens (e.g., blood, urine, swabs, etc.) are pooled and tested together; if the merged sample tests positive for infection, then each specimen from the pool is tested individually. Through this procedure, laboratories can reduce the expected number of tests required to screen the population, as individual tests are only carried out when the pooled test detects an infection. Several different partitions of the population can be used to form the pools. In this study, we analyze the performance of ordered partitions, those in which subjects with similar probability of infection are pooled together. We derive sufficient conditions under which ordered partitions outperform other types of partitions in terms of minimizing the expected number of tests, the expected number of false negatives, and the expected number of false positive classifications. These sufficient conditions can be easily verified in practical applications once the dilution effect has been estimated. We also propose a measure of equity and present conditions under which this measure is maximized by ordered partitions.

Evaluation of sample pooling for the detection of SARS-CoV-2 in a resource-limited setting, Dominican Republic.

Introduction: COVID-19 is a worldwide public health threat. Diagnosis by RT-PCR has been employed as the standard method to confirm viral infection. Sample pooling testing can optimize the resources by reducing the workload and reagents shortage, and be useful in laboratories and countries with limited resources. This study aims to evaluate SARS-CoV-2 detection by sample pooling testing in comparison with individual sample testing. Materials and methods: We created 210 pools out of 245 samples, varying from 4 to 10 samples per pool, each containing a positive sample. We conducted detection of SARS-CoV-2-specific RdRp/E target sites. Results: Pooling of three samples for SARS-CoV-2 detection might be an efficient strategy to perform without losing RT-PCR sensitivity. Conclusions: Considering the positivity rate in Dominican Republic and that larger sample pools have higher probabilities of obtaining false negative results, the optimal sample size to perform a pooling strategy shall be three samples.

Introducción: La COVID-19 es una amenaza de salud pública mundial. La RT-PCR es el método estándar para confirmar la infección. La estrategia de pruebas de muestras agrupadas puede reducir la carga de trabajo y la escasez de reactivos, y ser útil en países con escasos recursos. Evaluamos la detección del SARS-CoV-2 mediante esta estrategia en comparación con pruebas individuales. Materiales y métodos: Creamos 210 grupos de 245 muestras, de 4 a 10 muestras por grupo, cada uno con una muestra positiva. Realizamos extracción de ARN y qRT-PCR para detectar la presencia de la diana RdRp/E. Resultados: La combinación de hasta 3 muestras para la detección del SARS-CoV-2 podría ser una estrategia eficaz sin perder la sensibilidad. Conclusiones: Considerando la tasa de positividad en República Dominicana y que los grupos con más muestras tienen mayor probabilidad de obtener resultados falsos negativos, el tamaño óptimo para realizar esta estrategia es de 3 muestras.

Diagnostic performance of RT-PCR-based sample pooling strategy for the detection of SARS-CoV-2.

BACKGROUND: The rapid spread of SARS-CoV-2 has created a shortage of supplies of reagents for its detection throughout the world, especially in Latin America. The pooling of samples consists of combining individual patient samples in a block and analyzing the group as a particular sample. This strategy has been shown to reduce the burden of laboratory material and logistical resources by up to 80%. Therefore, we aimed to evaluate the diagnostic performance of the pool of samples analyzed by RT-PCR to detect SARS-CoV-2. METHODS: A cross-sectional study of diagnostic tests was carried out. We individually evaluated 420 samples, and 42 clusters were formed, each one with ten samples. These clusters could contain 0, 1 or 2 positive samples to simulate a positivity of 0, 10 and 20%, respectively. RT-PCR analyzed the groups for the detection of SARS-CoV-2. The area under the ROC curve (AUC), the Youden index, the global and subgroup sensitivity and specificity were calculated according to their Ct values that were classified as high (H: ≤ 25), moderate (M: 26-30) and low (L: 31-35) concentration of viral RNA. RESULTS: From a total of 42 pools, 41 (97.6%) obtained the same result as the samples they contained (positive or negative). The AUC for pooling, Youden index, sensitivity, and specificity were 0.98 (95% CI, 0.95-1); 0.97 (95% CI, 0.90-1.03); 96.67% (95% CI; 88.58-100%) and 100% (95% CI; 95.83-100%) respectively. In the stratified analysis of the pools containing samples with Ct ≤ 25, the sensitivity was 100% (95% CI; 90-100%), while with the pools containing samples with Ct ≥ 31, the sensitivity was 80% (95% CI, 34.94-100%). Finally, a higher median was observed in the Ct of the clusters, with respect to the individual samples (p < 0.001). CONCLUSIONS: The strategy of pooling nasopharyngeal swab samples for analysis by SARS-CoV-2 RT-PCR showed high diagnostic performance.

The effect of correlation and false negatives in pool testing strategies for COVID-19.

During the current COVID-19 pandemic, active testing has risen as a key component of many response strategies around the globe. Such strategies have a common denominator: the limited availability of diagnostic tests. In this context, pool testing strategies have emerged as a means to increase testing capacity. The efficiency gains obtained by using pool testing, derived from testing combined samples simultaneously, vary according to the spread of the SARS-CoV-2 virus in the population being tested. Motivated by the need for testing closed populations, such as long-term care facilities (LTCFs), where significant correlation in infections is expected, we develop a probabilistic model for settings where the test results are correlated, which we use to compute optimal pool sizes in the context of two-stage pool testing schemes. The proposed model incorporates the specificity and sensitivity of the test, which makes it possible to study the impact of these measures on both the expected number of tests required for diagnosing a population and the expected number and variance of false negatives. We use our experience implementing pool testing in LTCFs managed by SENAMA (Chile's National Service for the Elderly) to develop a simulation model of contagion dynamics inside LTCFs, which incorporates testing and quarantine policies implemented by SENAMA. We use this simulation to estimate the correlation of test results among collected samples when following SENAMA's testing guidelines. Our results show that correlation estimates are high in settings representative of LTCFs, which validates the use of the proposed model for incorporating correlation in determining optimal pool sizes for pool testing strategies. Generally, our results show that settings in which pool testing achieves efficiency gains, relative to individual testing, are likely to be found in practice. Moreover, the results show that incorporating correlation in the analysis of pool testing strategies both improves the expected efficiency and broadens the settings in which the technique is preferred over individual testing.

A discussion on the minimum required number of tests in two common pooling test methods for SARS-CoV-2.

Pooling of samples in detecting the presence of virus is an effective and efficient strategy in screening carriers in a large population with low infection rate, leading to reduction in cost and time. There are a number of pooling test methods, some being simple and others being complicated. In such pooling tests, the most important parameter to decide is the pool or group size, which can be optimised mathematically. Two pooling methods are relatively simple. The minimum numbers required in these two tests for a population with known infection rate are discussed and compared. Results are useful for identifying asymptomatic carriers in a short time and in implementing health codes systems.

Feasibility, efficiency & effectiveness of pooled sample testing strategy (pooled NAAT) for molecular testing of COVID-19.

BACKGROUND & OBJECTIVES: During the current COVID-19 pandemic, a large number of clinical samples were tested by real-time PCR. Pooling the clinical samples before testing can be a good cost-saving and rapid alternative for screening large populations. The aim of this study was to compare the performance characteristics, feasibility and effectiveness of pooling nasal swab and throat swab samples for screening and diagnosis of SARS-CoV-2. METHODS: The pool testing was applied on a set of samples coming from low COVID-19 positivity areas. A total of 2410 samples were tested in pools of five samples each. A total of five pools of five samples each were generated and tested for E gene. RESULTS: Of the total of 482 pools (2410 samples) 24 pools flagged positive. Later on pool de-convolution, a total of 26 samples were detected as positive for COVID-19, leading to positivity of about one per cent in the test population. For the diagnosis of individual samples, the pooling strategies resulted in cost savings of 75 per cent (5 samples per pool). INTERPRETATION & CONCLUSIONS: It was observed that testing samples for COVID-19 by reverse transcription (RT)- PCR after pooling could be a cost-effective method which would save both in manpower and cost especially for resource-poor countries and at a time when test kits were short in supply.

Optimization of group size in pool testing strategy for SARS-CoV-2: A simple mathematical model.

Coronavirus disease (Covid-19) has reached unprecedented pandemic levels and is affecting almost every country in the world. Ramping up the testing capacity of a country supposes an essential public health response to this new outbreak. A pool testing strategy where multiple samples are tested in a single reverse transcriptase-polymerase chain reaction (RT-PCR) kit could potentially increase a country's testing capacity. The aim of this study is to propose a simple mathematical model to estimate the optimum number of pooled samples according to the relative prevalence of positive tests in a particular healthcare context, assuming that if a group tests negative, no further testing is done whereas if a group tests positive, all the subjects of the group are retested individually. The model predicts group sizes that range from 11 to 3 subjects. For a prevalence of 10% of positive tests, 40.6% of tests can be saved using testing groups of four subjects. For a 20% prevalence, 17.9% of tests can be saved using groups of three subjects. For higher prevalences, the strategy flattens and loses effectiveness. Pool testing individuals for severe acute respiratory syndrome coronavirus 2 is a valuable strategy that could considerably boost a country's testing capacity. However, further studies are needed to address how large these groups can be, without losing sensitivity on the RT-PCR. The strategy best works in settings with a low prevalence of positive tests. It is best implemented in subgroups with low clinical suspicion. The model can be adapted to specific prevalences, generating a tailored to the context implementation of the pool testing strategy.

Rapid, large-scale, and effective detection of COVID-19 via non-adaptive testing.

Pooling of samples can increase lab capacity when using Polymerase chain reaction (PCR) to detect diseases such as COVID-19. However, pool testing is typically performed via an adaptive testing strategy which requires a feedback loop in the lab and at least two PCR runs to confirm positive results. This can cost precious time. We discuss a non-adaptive testing method where each sample is distributed in a prescribed manner over several pools, and which yields reliable results after one round of testing. More precisely, assuming knowledge about the overall incidence rate, we calculate explicit error bounds on the number of false positives which scale favourably with pool size and sample multiplicity. This allows for hugely streamlined PCR testing and cuts in detection times for a large-scale testing scenario. A viable consequence of this method could be real-time screening of entire communities, frontline healthcare workers and international flight passengers, for example, using the PCR machines currently in operation.

Modelling pool testing for SARS-CoV-2: addressing heterogeneity in populations.

Amplifying the testing capacity and making better use of testing resources is a crucial measure when fighting any pandemic. A pooled testing strategy for SARS-CoV-2 has theoretically been shown to increase the testing capacity of a country, especially when applied in low prevalence settings. Experimental studies have shown that the sensitivity of reverse transcription-polymerase chain reaction is not affected when implemented in small groups. Previous models estimated the optimum group size as a function of the historical prevalence; however, this implies a homogeneous distribution of the disease within the population. This study aimed to explore whether separating individuals by age groups when pooling samples results in any further savings on test kits or affects the optimum group size estimation compared to Dorfman's pooling, based on historical prevalence. For this evaluation, age groups of interest were defined as 0-19 years, 20-59 years and over 60 years old. Generalisation of Dorfman's pooling was performed by adding statistical weight to the age groups based on the number of confirmed cases and tests performed in the segment. The findings showed that when the pooling samples are based on age groups, there is a decrease in the number of tests per subject needed to diagnose one subject. Although this decrease is minuscule, it might account for considerable savings when applied on a large scale. In addition, the savings are considerably higher in settings where there is a high standard deviation among the positivity rate of the age segments of the general population.

Improved GNSS Localization and Byzantine Detection in UAV Swarms.

Many tasks performed by swarms of unmanned aerial vehicles require localization. In many cases, the sensors that take part in the localization process suffer from inherent measurement errors. This problem is amplified when disruptions are added, either endogenously through Byzantine failures of agents within the swarm, or exogenously by some external source, such as a GNSS jammer. In this paper, we first introduce an improved localization method based on distance observation. Then, we devise schemes for detecting Byzantine agents, in scenarios of endogenous disruptions, and for detecting a disrupted area, in case the source of the problem is exogenous. Finally, we apply pool testing techniques to reduce the communication traffic and the computation time of our schemes. The optimal pool size should be chosen carefully, as very small or very large pools may impair the ability to identify the source/s of disruption. A set of simulated experiments demonstrates the effectiveness of our proposed methods, which enable reliable error estimation even amid disruptions. This work is the first, to the best of our knowledge, that embeds identification of endogenous and exogenous disruptions into the localization process.

Reemergence of St. Louis Encephalitis Virus, California, 2015.

St. Louis encephalitis virus infection was detected in summer 2015 in southern California after an 11-year absence, concomitant with an Arizona outbreak. Sequence comparisons showed close identity of California and Arizona isolates with 2005 Argentine isolates, suggesting introduction from South America and underscoring the value of continued arbovirus surveillance.

A general regression framework for group testing data, which incorporates pool dilution effects.

Group testing, through the use of pooling, has been widely implemented as a more efficient means to screen individuals for infectious diseases. Typically, in these settings, practitioners are tasked with the complimentary goals of both case identification and estimation. For these purposes, many group testing strategies have been proposed, which address issues such as preserving anonymity in estimation studies, quality control, and classification. In general, these strategies require that a significant number of the individuals be retested, either in pools or individually. In order to provide practitioners with a general methodology that can be used to accurately and precisely analyze data of this form, herein, we propose a binary regression framework that can incorporate data arising from any group testing strategy. Further, we relax previously made assumptions regarding testing error rates by relating the diagnostic testing results to the latent biological marker levels of the individuals being tested. We investigate the finite sample performance of our proposed methodology through simulation and by applying our techniques to hepatitis B data collected as part of a study involving Irish prisoners.

Universal and Expanded Screening Strategy for Congenital Cytomegalovirus Infection: Is Pool Testing by a Rapid Molecular Test in Saliva a New Choice in Developing Countries?

BACKGROUND: Several screening strategies for identifying congenital CMV (cCMV) have been proposed; however, the optimal solution has yet to be determined. We aimed to determine the prevalence of cCMV by universal screening with saliva pool testing and to identify the clinical variables associated with a higher risk of cCMV to optimize an expanded screening strategy. METHODS: We carried out a prospective universal cCMV screening (September/2022 to August/2023) of 2186 newborns, analyzing saliva samples in pools of five (Alethia-LAMP-CMV®) and then performed confirmatory urine CMV RT-PCR. Infants with risk factors (small for gestational age, failed hearing screening, HIV-exposed, born to immunosuppressed mothers, or <1000 g birth weight) underwent expanded screening. Multivariate analyses were used to assess the association with maternal/neonatal variables. RESULTS: We identified 10 infants with cCMV (prevalence: 0.46%, 95% CI 0.22-0.84), with significantly higher rates (2.1%, 95% CI 0.58-5.3) in the high-risk group (p = 0.04). False positives occurred in 0.09% of cases. No significant differences in maternal/neonatal characteristics were observed, except for a higher prevalence among infants born to non-Chilean mothers (p = 0.034), notably those born to Haitian mothers (1.5%, 95% CI 0.31-4.34), who had higher odds of cCMV (OR 6.82, 95% CI 1.23-37.9, p = 0.04). Incorporating maternal nationality improved predictive accuracy (AUC: 0.65 to 0.83). CONCLUSIONS: For low-prevalence diseases such as cCMV, universal screening with pool testing in saliva represents an optimal and cost-effective approach to enhance diagnosis in asymptomatic patients. An expanded screening strategy considering maternal nationality could be beneficial in resource-limited settings.

Effects of infection control measures towards preventing SARS-CoV-2 outbreaks in a German choir boarding school from March 2020 to April 2022.

Background: Singing in a choir was associated with larger outbreaks in the beginning of the SARS-CoV-2 pandemic. Materials and methods: We report on the effect and acceptance of various infection control measures on the occurrence of SARS-CoV-2 infections in the world famous Domspatzen boys' choir from March 2020 to April 2022. Results: In addition to basic general hygiene measures, systematic rRT-PCR testing and scientifically approved concepts of distancing during singing were applied. While single infections of choir members could not be avoided, singing-related outbreaks were not observed. Until the Omicron variant emerged, potential transmission of SARS-CoV-2 in the school was limited to only one case. Incidences at the school were never higher than in the comparable general population until then. While the impact of the pandemic on daily life and singing was rated as severe, especially by staff members, most students agreed with the usefulness of protection measures and rated them as acceptable. Students viewed regular testing as the most important tool to increase safety in the school. Discussion: A bundle of infection control measures including regular testing can prevent outbreaks of SARS-CoV-2 even in the setting of choir singing. Measures are acceptable for choir members if they allow to continue with singing and performing.

Swab pooling enables rapid expansion of high-throughput capacity for SARS-CoV-2 community testing.

BACKGROUND: The challenges of rapid upscaling of testing capacity were a major lesson from the COVID-19 pandemic response. The need for process adjustments in high-throughput testing laboratories made sample pooling a challenging option to implement. OBJECTIVE: This study aimed to evaluate whether pooling samples at source (swab pooling) was as effective as qRT-PCR testing of individuals in identifying cases of SARS-CoV-2 in real-world community testing conditions using the same high-throughput pipeline. METHODS: Two cohorts of 10 (Pool10: 1,030 participants and 103 pools) and 6 (Pool6: 1,284 participants and 214 pools) samples per pool were tested for concordance, sensitivity, specificity, and Ct value differences with individual testing as reference. RESULTS: Swab pooling allowed unmodified application of an existing high-throughput SARS-Cov-2 testing pipeline with only marginal loss of accuracy. For Pool10, concordance was 98.1% (95% Confidence interval: 93.3-99.8%), sensitivity was 95.7% (85.5-99.5%), and specificity was 100.0% (93.6-100.0%). For Pool6, concordance was 97.2% (94.0-99.0%), sensitivity was 97.5% (93.7-99.3%), and specificity was 96.4% (87.7-99.6%). Differences of outcomes measure between pool size were not significant. Most positive individual samples, which were not detected in pools, had very low viral concentration. If only individual samples with a viral concentration > 400 copies/ml (i.e. Ct value < 30) were considered positive, the overall sensitivity of pooling increased to 99.5%. CONCLUSION: The study demonstrated high sensitivity and specificity by swab pooling and the immediate capability of high-throughput laboratories to implement this method making it an option in planning of rapid upscaling of laboratory capacity for future pandemics.

Evaluation of sample pooling for the detection of SARS-CoV-2 in a resource-limited setting, Dominican Republic.

INTRODUCTION: COVID-19 is a worldwide public health threat. Diagnosis by RT-PCR has been employed as the standard method to confirm viral infection. Sample pooling testing can optimize the resources by reducing the workload and reagents shortage, and be useful in laboratories and countries with limited resources. This study aims to evaluate SARS-CoV-2 detection by sample pooling testing in comparison with individual sample testing. MATERIALS AND METHODS: We created 210 pools out of 245 samples, varying from 4 to 10 samples per pool, each containing a positive sample. We conducted detection of SARS-CoV-2-specific RdRp/E target sites. RESULTS: Pooling of three samples for SARS-CoV-2 detection might be an efficient strategy to perform without losing RT-PCR sensitivity. CONCLUSIONS: Considering the positivity rate in Dominican Republic and that larger sample pools have higher probabilities of obtaining false negative results, the optimal sample size to perform a pooling strategy shall be three samples.

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR.

Introduction: The declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance. Methods: Using 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples. Results: The study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes. Discussion: The employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method's robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method's independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves.

Effectiveness Of Pcr Pool Testing For Screening Of Covid-19 Infection In Pakistan.

BACKGROUND: We tested the utility of mini-pool PCR testing for the rational use of PCR consumables in screening for CoViD-19. METHODS: After pilot experiments, 3-samples pool size was selected. One step RT-PCR was performed. The samples in the mini-pool having COVID gene amplification were tested individually. RESULTS: 1548 samples tested in 516 mini-pools resulted 396 mini-pools as negative and 120 as positive. Upon individual testing, 110 samples tested positive and 9 were inconclusive. 876 PCR reactions were performed to test 1548 samples, saving 43% PCR reagents. Centres with low prevalence resulted in most saving on reagents (50%), while centres with high prevalence resulted in more test reactions. Testing of individual samples resulted in delays in reporting. CONCLUSIONS: Pooling can increase lab capacity, however, pooling delays results and cause degradation of samples.

Pool Testing for COVID-19: Suitable Splitting Procedure and Pool Size for India.

OBJECTIVE: Coronavirus disease (COVID-19) has emerged as a global pandemic for public health due to the large scale outbreak, therefore there is an urgent need to detect the infected cases quickly and isolate them in order to suppress the further spread of the disease. This study tries to identify a suitable pool testing method and algorithm for COVID-19. METHODS: This study tries to derive a general equation for the number of tests required for a pooled sample to detect every infected individual in the specific pool. The gain in pool testing over the normal procedure is quantified by the percentage of tests required compared to individual testing. RESULTS: The percentage of tests required by the pool testing strategy varies according to the different splitting procedures, the size of the pooled sample, and the probability of an individual being infected in the population. If the probability of infection is 0.05, then for a pool size of 32, only 14 tests are sufficient to detect every infected individual. CONCLUSION: The number of tests required to detect infected individuals by using the pooling method is much lower than individual testing. This may help us with increasing our testing capacity for COVID-19 by testing a large number of individuals in less time with limited resources.