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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.
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Infecções por Citomegalovirus , Citomegalovirus , Países em Desenvolvimento , Triagem Neonatal , Saliva , Humanos , Saliva/virologia , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/epidemiologia , Infecções por Citomegalovirus/virologia , Recém-Nascido , Feminino , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Estudos Prospectivos , Triagem Neonatal/métodos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Prevalência , Programas de Rastreamento/métodos , Sensibilidade e Especificidade , Gravidez , Fatores de RiscoRESUMO
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%.
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Infecções por Citomegalovirus , Citomegalovirus , Lactente , Humanos , Recém-Nascido , Citomegalovirus/genética , Saliva , Infecções por Citomegalovirus/diagnóstico , Triagem Neonatal/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
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.
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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.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , República Dominicana , Região de Recursos Limitados , Manejo de Espécimes/métodosRESUMO
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.
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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.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiologia , Estudos Retrospectivos , Reação em Cadeia da Polimerase , Teste para COVID-19RESUMO
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.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Estudos Transversais , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genéticaRESUMO
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.
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COVID-19 , Idoso , COVID-19/diagnóstico , Humanos , Modelos Estatísticos , Pandemias , SARS-CoV-2RESUMO
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.
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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Adulto JovemRESUMO
Abstract INTRODUCTION: The pool testing technique optimizes the number of tests performed and reduces the delivery time of results, which is an interesting strategy for the health crisis caused by the COVID-19 pandemic. This integrative review investigated studies in which pool testing was carried out for epidemiological or screening purposes to analyze its clinical or cost effectiveness and assessed the applicability of this method in high-, middle-, and low-income countries. METHODS: This integrative review used primary studies published in the MEDLINE, EMBASE, Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), and Cochrane Library databases. RESULTS: A total of 435 studies were identified: 35.3% were carried out in Asia, 29.4% in Europe, 29.4% in North America, and 5.9% in Oceania. CONCLUSIONS: This review suggests that pool testing in the general population may be a useful surveillance strategy to detect new variants of SARS-CoV-2 and to evaluate the period of immunogenicity and global immunity from vaccines.
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Humanos , SARS-CoV-2 , COVID-19 , Programas de Rastreamento , Pandemias , Teste para COVID-19RESUMO
Abstract The global shortage of reagents and kits for nucleic acid extraction and molecular detection of SARS-CoV-2 requires new cost-effective strategies for the diagnosis of suspected COVID-19 cases, especially in countries that need to increase detection capacity. Pooled nucleic acid testing has been extensively used as a cost-effective strategy for HIV, HepB, HepC and influenza. Also, protocols dispensing of RNA extraction appears as an attractive option for detection of SARS-CoV-2. In this study, we found that pooling of 5 samples showed that CT variations were in the range of 1.0-4,5 units, with less likelihood of a false negative result. Results of the sample without nucleic acid ex-traction, was unsatisfactory, with a significant increase in CT values, and thus for risk of a false negative result. In conclusion, pooling nasopharyngeal samples with both automated and manual extraction proved reliable, and thus a potential efficient alternative for the diagnosis of suspected COVID-19 in developing countries.
Resumen La escasez mundial de reactivos para la extracción de ácidos nucleicos y la detección molecular de SARS-CoV-2 requiere de nuevas estrategias de mayor rendimiento para el diagnóstico de casos sospechosos de COVID-19, especialmente en países que necesitan aumentar su capacidad diagnóstica. La detección de ácidos nucleicos en muestras agrupadas o pool testing se ha utilizado ampliamente como una estrategia costo-efectiva para el VIH, hepatitis B, hepatitis C e influenza. Adicionalmente, los protocolos que no requieren extracción de ARN aparecen como una opción para la detección de SARS-CoV-2. En este trabajo, presentamos los resultados de una estrategia detección de SARS-CoV-2 en muestras agrupadas, que incluye diferentes métodos de extracción de ARN que puede ser una estrategia atractiva para los países en desarrollo. La agrupación de 5 muestras mostró variaciones CT en el rango de 1,0 a 4,5 unidades, con una baja probabilidad de obtener falsos negativos, a diferencias de los resultados agregando muestras agrupadas directamente en la reacción de amplificación de SARS-CoV-2. En conclusión, la agrupación de muestras nasofaríngeas, demostró ser un método confiable y, por lo tanto, una alternativa para aumentar el rendimiento en el diagnóstico de COVID-19 para países en desarrollo.
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Humanos , Pneumonia Viral/diagnóstico , Infecções por Coronavirus/diagnóstico , Pandemias , RNA Viral , Técnicas de Laboratório Clínico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Países em DesenvolvimentoRESUMO
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.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Programas de Rastreamento/métodos , Modelos Teóricos , COVID-19/epidemiologia , Humanos , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.