Assessment of Nucleic Acid Extraction Kits for SARS-CoV-2 Surveillance in Wastewater Samples

Mersin Üniversitesi Tıp Fakültesi Lokman Hekim Tıp Tarihi ve Folklorik Tıp Dergisi, Journal Year: 2025, Volume and Issue: 15(1), P. 241 - 251

Published: Jan. 29, 2025

Objective: The aim of this study is to evaluate the effectiveness three commercial nucleic acid extraction kits (kit A, B and C) in isolating SARS-CoV-2 viral RNA from wastewater samples. Method: In study, water samples were collected March 2021 treatment plants located different parts Istanbul, it was confirmed that they negative for SARS-CoV-2. Different concentrations virus, previously inactivated at BSL-3 laboratory Pendik Veterinary Control Institute, added quantification performed using RT-qPCR kit specific Results: At end determined C yielded highest total produced more consistent results, significantly outperforming other two terms yield purity. Statistical analysis revealed significant differences (p < 0.05) gene copy numbers 0.01) between kits, demonstrated superior linearity reproducibility. Conclusion: According findings, although all evaluated are suitable detecting samples, provides most efficient reliable performance, especially high-throughput studies. Additionally, highlights importance selecting appropriate methods surveillance, which serves as an early warning system outbreaks threaten public health.

Language: Английский

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Evaluating Interlaboratory Variability in Wastewater-Based COVID-19 Surveillance

Microorganisms, Journal Year: 2025, Volume and Issue: 13(3), P. 526 - 526

Published: Feb. 27, 2025

Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking have been developed globally, underscoring need interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories network in wastewater samples across Lombardy region (Italy). The aimed evaluate reliability identify potential sources variability using robust statistical approaches. Three were analyzed parallel by four identical pre-analytical (PEG-8000-based centrifugation) analytical processes (qPCR targeting N1/N3 Orf-1ab). A two-way ANOVA framework Generalized Linear Models applied, multiple pairwise performed Bonferroni post hoc test. analysis revealed that primary source results associated with phase. This likely influenced differences standard curves used quantify concentrations, as well size treatment plants. findings this study highlight importance testing verifying determinations identifying key variation.

Language: Английский

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EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond

Human Genomics, Journal Year: 2024, Volume and Issue: 18(1)

Published: June 27, 2024

Abstract Background Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens contaminants. To achieve thorough comprehension of present upcoming practices identify challenges opportunities standardisation improvement WWS methodologies, two EU surveys were conducted targeting over 750 laboratories across Europe other regions. The first survey explored diverse range activities currently undertaken or planned laboratories. second specifically targeted methods quality controls utilised SARS-CoV-2 surveillance. Results findings the provide comprehensive insight into procedures methodologies applied WWS. In Europe, primarily focuses on with 99% participants dedicated this virus. However, responses highlighted lack employed SARS-CoV-2. pathogens, including antimicrobial resistance, is fragmented only limited number Notably, these are anticipated expand future. Survey replies emphasise collective recognition need enhance accuracy results practices, reflecting shared commitment advancing precision effectiveness methodologies. Conclusions These identified standardised common standards reference materials reliability addition, it important broaden efforts beyond include emerging resistance ensure approach protecting health.

Language: Английский

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Evaluating the sensitivity of droplet digital PCR for the quantification of SARS-CoV-2 in wastewater

Frontiers in Public Health, Journal Year: 2023, Volume and Issue: 11

Published: Dec. 27, 2023

Wastewater surveillance for SARS-CoV-2 has been demonstrated to be a valuable tool in monitoring community-level virus circulation and assessing new outbreaks. It may become useful the early detection response future pandemics, enabling public health authorities implement timely interventions mitigate spread of infectious diseases with fecal excretion their agents. also offers chance cost-effective surveillance. Reverse transcription-quantitative polymerase chain reaction (RTqPCR) is most commonly used method viral RNA wastewater due its sensitivity, reliability, widespread availability. However, recent studies have indicated that reverse transcription droplet digital PCR (RTddPCR) potential offer improved sensitivity accuracy quantifying samples. In this study, we compared performance RTqPCR RTddPCR approaches quantification on samples collected during third epidemic wave Saxony, Germany, characterized by low-incidence infection periods. The determined limits (LOD) (LOQ) were within same order magnitude, no significant differences observed between respect number positive or quantifiable Our results indicate both are highly sensitive methods detecting SARS-CoV-2. Consequently, actual gain associated ddPCR lags behind theoretical expectations. Hence, choice two further environmental programs rather matter available resources throughput requirements.

Language: Английский

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Influence of storage conditions and multiple freeze-thaw cycles on N1 SARS-CoV-2, PMMoV, and BCoV signal

The Science of The Total Environment, Journal Year: 2023, Volume and Issue: 896, P. 165098 - 165098

Published: June 29, 2023

Language: Английский

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Clinical evaluation of a multiplex droplet digital PCR for pathogen detection in critically ill COVID-19 patients with bloodstream infections

Infection, Journal Year: 2023, Volume and Issue: 52(3), P. 1027 - 1039

Published: Dec. 21, 2023

Abstract Background Nosocomial bloodstream infections (nBSI) have emerged as a clinical concern for physicians treating COVID-19 patients. In this study, we aimed to evaluate the effectiveness of multiplex ddPCR in detecting bacterial pathogens blood critically ill Methods This prospective diagnostic study included RT-PCR-confirmed patients admitted our hospital from December 2022 February 2023. A assay was used detect common and AMR genes samples patients, along with antimicrobial susceptibility testing (AST). The performance evaluated by comparing results those obtained through culture diagnosis. Additionally, ability resistance compared AST results. Results Of 200 collected 184 45 (22.5%) were positive using culture, while 113 (56.5%) targets assay. outperformed pathogen detection rate, mixed infection fungal rate. Acinetobacter baumannii Klebsiella pneumoniae most commonly detected followed Enterococcus Streptococcus . Compared achieved sensitivity 75.5%, specificity 51.0%, PPV 30.9%, NPV 87.8%, respectively. However, there significant differences among different species, where Gram-negative bacteria highest 90.3%. When on ground diagnosis, sensitivity, specificity, 78.1%, 90.5%, 94.7%, 65.5%, addition, 23 cases bla KPC , which shown better consistent test than other genes. few detected, indicating that application gene limited. Conclusion had significantly higher positivity could be an effective tool BSIs improve patient outcomes reduce burden sepsis healthcare system, though is room optimization panels used.- Adjusting include E. faecalis faecium well Candida albicans glabrata ddPCR' s effectiveness. further research needed explore potential predicting detection.

Language: Английский

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Presence, transmission, and management of the SARS-CoV-2 in wastewater: a brief review

International Journal of Environmental Science and Technology, Journal Year: 2024, Volume and Issue: 21(15), P. 9719 - 9742

Published: May 12, 2024

Language: Английский

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Comparison of Different Reverse Transcriptase–Polymerase Chain Reaction–Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study

JMIR Public Health and Surveillance, Journal Year: 2024, Volume and Issue: 10, P. e53175 - e53175

Published: Aug. 19, 2024

Background Many countries have applied the wastewater surveillance of COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in are quantitative reverse transcriptase–polymerase chain reaction (RT-qPCR) and transcriptase–droplet digital polymerase (RT-ddPCR). Previous comparison studies produced conflicting results, thus more research on subject is required. Objective This study aims compare RT-qPCR RT-ddPCR wastewater. It also aimed investigate effect changes analytical pipeline, including RNA extraction kit, RT-PCR target gene assay, results. Another aim was find a detection method low-resource settings. Methods We compared 2 kits, TaqMan QuantiTect RT-qPCR, based sensitivity, positivity rates, variability, correlation copy numbers incidence COVID-19. Furthermore, we methods, column- magnetic-bead–based. In addition, assessed assays N1 N2, ddPCR E. Reverse transcription strand invasion-based amplification (RT-SIBA) detect from qualitatively. Results Our results indicated that sensitive RT-ddPCR. had highest rate (26/30), its limit lowest (0.06 copies/µL). However, obtained best between number using (correlation coefficient [CC]=0.697, P<.001). found significant difference sensitivity kit first having significantly lower higher than latter. assay both while no targets use different kits affected result when used. RT-SIBA able Conclusions As our study, as well previous studies, has shown be should considered, especially if amount circulating population low. All analysis steps must optimized showed all compatibility extraction, influence could qualitative sufficient.

Language: Английский

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Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring

The Science of The Total Environment, Journal Year: 2023, Volume and Issue: 903, P. 166540 - 166540

Published: Aug. 25, 2023

Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used early warning system for monitoring the emergence of novel pathogens or viral variants. However, a timely transmission results, sophisticated sample logistics and analytics performed in decentralized laboratories close sampling sites are required. Since multiple commonly use custom in-house workflows purification PCR-analysis, comparative quality control analytical procedures is essential report reliable comparable results. In this study, we interlaboratory comparison at specialized PCR high-throughput-sequencing (HTS)-based analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic variants graduated concentrations ratios. Samples sent participating analysis using laboratory specific methods reported genome copy numbers detection compared expected values. All PCR-laboratories equivalents (GCE) all mean intra- inter-laboratory variability 19 % 104 %, respectively, largely reproducing spike-in scheme. PCR-based genotyping was, dependence underlying PCR-assay performance, able predict relative amount variant substitutions even amount. The identification by HTS, however, required >100 copies/ml wastewater had limited predictive value when analyzing coverage below 60 %. This test demonstrates that despite highly heterogeneous isolation procedures, overall GCE mutations determined accurately. Hence, feasible generate since not assays detected correct variant, prior evaluation sequencing well sustained such comparisons mandatory detection.

Language: Английский

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SARS-CoV-2 Wastewater Monitoring in Thuringia, Germany: Analytical Aspects and Normalization of Results

Water, Journal Year: 2023, Volume and Issue: 15(24), P. 4290 - 4290

Published: Dec. 15, 2023

Wastewater monitoring for SARS-CoV-2 is a valuable tool surveillance in public health. However, reliable analytical methods and appropriate approaches the normalization of results are important requirements implementing state-wide programs. In times insufficient case reporting, evaluation wastewater data challenging. Between December 2021 July 2022, we analyzed 646 samples from 23 WWTPs Thuringia, Germany. We investigated performance direct capture-based method RNA extraction (4S-method) evaluated four (NH4-N, COD, Ntot, PMMoV) pooled analysis using different epidemiological metrics. The 4S were well met. could be successfully applied to implement program including large number medium small treatment plants (<100,000 p.e) high spatial density. Correlations between 7-day incidence or 7-day-hospitalization strong independent method. For test positivity rate, PMMoV-normalized showed better correlation than normalized with chemical markers. low testing frequency 7-day-incidence might become less reliable. Alternative metrics like hospital admissions increasingly evaluating methods. Furthermore, future studies need address variance biological replicates wastewater.

Language: Английский

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