Evaluation of droplet digital qRT-PCR (dd qRT-PCR) for quantification of SARS CoV-2 RNA in stool and urine specimens of COVID-19 patients

Shinde, Manohar; Lavania, Mallika; Rawal, J; Chavan, Nutan; Shinde, Pooja

doi:10.3389/fmed.2023.1148688

Cited by 2 publications

(1 citation statement)

References 27 publications

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“…Digital PCR’s ability to detect different types of viruses, including respiratory pathogens and others like Ebola and Dengue, in blood samples, has been validated ( 34 , 35 ). For instance, our work on the detection of SARS-CoV-2 in saliva ( 26 ), and the effectiveness of digital PCR in other matrices such as urine and feces for SARS-CoV-2 ( 36 ) and bacteria in blood ( 34 ), underlines the flexibility of this technology in a variety of diagnostic scenarios.…”

Section: Discussionmentioning

confidence: 99%

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

Pacini,

Paredes,

Heckel

et al. 2024

Front. Public Health

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IntroductionThe 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.MethodsUsing 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.ResultsThematic analysis of the interviews identified three major challenges faced by public health officials in promoting vaccination: dissemination of misinformation in media, political polarization of COVID and its contribution to vaccine acceptance and COVID fatigue, and assessment of the risks associated with disease severity versus vaccine safety considering limited public health resources.DiscussionThe 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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Section: Discussionmentioning

confidence: 99%

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

Pacini,

Paredes,

Heckel

et al. 2024

Front. Public Health

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Multisite community-scale monitoring of respiratory and enteric viruses in the effluent of a nursing home and in the inlet of the local wastewater treatment plant

Manoha,

Dequiedt,

Thery

et al. 2024

Appl Environ Microbiol

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The aim of this study was to evaluate whether community-level monitoring of respiratory and enteric viruses in wastewater can provide a comprehensive picture of local virus circulation. Wastewater samples were collected weekly at the wastewater treatment plant (WWTP) inlet and at the outlet of a nearby nursing home (NH) in Burgundy, France, during the winter period of 2022/2023. We searched for the pepper mild mottle virus as an indicator of fecal content as well as for the main respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus] and enteric viruses (rotavirus, sapovirus, norovirus, astrovirus, and adenovirus). Samples were analyzed using real-time reverse transcription PCR-based methods. SARS-CoV-2 was the most frequently detected respiratory virus, with 66.7% of positive samples from the WWTP and 28.6% from the NH. Peaks of SARS-CoV-2 were consistent with the chronological incidence of infections recorded in the sentinel surveillance and the nearby hospital databases. The number of positive samples was lower in the NH than in WWTP for the three respiratory viruses. Enteric viruses were frequently detected, most often sapovirus and norovirus genogroup II, accounting both for 77.8% of positive samples in the WWTP and 57.1% and 37%, respectively, in the NH. The large circulation of sapovirus was unexpected in particular in the NH. Combined wastewater surveillance using simple optimized methods can be a valuable tool for monitoring viral circulation and may serve as a suitable early warning system for identifying both local outbreaks and the onset of epidemics. These results encourage the application of wastewater-based surveillance (WBS) to SARS-CoV2, norovirus, and sapovirus. IMPORTANCE WBS provides valuable information on the spread of epidemic viruses in the environment using appropriate and sensitive detection methods. By monitoring the circulation of viruses using reverse transcription PCR methods in wastewater from the inlet of a wastewater treatment plant and the outlet of a nearby retirement home (connected to the same collective sewer network), we aimed to demonstrate that implementing combined WBS at key community sites allows effective detection of the occurrence of respiratory (influenza, respiratory syncytial virus, and SARS-CoV-2) and enteric (norovirus, rotavirus, and sapovirus) virus infections within a given population. This analysis on a localized scale provided new information on the viral circulation in the two different sites. Implementing WBS to monitor the circulation or the emergence of infectious diseases is an important means of alerting the authorities and improving public health management. WBS could participate actively to the health of humans, animals, and the environment.

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Evaluation of droplet digital qRT-PCR (dd qRT-PCR) for quantification of SARS CoV-2 RNA in stool and urine specimens of COVID-19 patients

Cited by 2 publications

References 27 publications

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

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

Multisite community-scale monitoring of respiratory and enteric viruses in the effluent of a nursing home and in the inlet of the local wastewater treatment plant

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