Comparison of Eleven RNA Extraction Methods for Poliovirus Direct Molecular Detection in Stool Samples

Akello, Joyce Odeke; Bujaki, Erika; Shaw, Alexander; Khurshid, Adnan; Arshad, Yasir; Troman, Catherine; Majumdar, Manasi; O’Toole, Áine; Rambaut, Andrew; Alam, Muhammad Masroor; Martı́n, Javier; Grassly, Nicholas C.

doi:10.1128/spectrum.04252-22

Cited by 4 publications

(4 citation statements)

References 25 publications

(34 reference statements)

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“…Chloroform-treated stool supernatant underwent RNA extraction using either QIAamp Viral RNA Mini Kits (#51106, used for samples processed August 2021 to September 2021) or Roche High Pure Viral RNA Kits (#11858882001, used for samples processed October 2021 to February 2022) according to manufacturers’ protocols using a volume of 140 µl and 200 µl of supernatant, respectively. RNA extraction kit selection was determined by kit availability, but both kits have been validated for the DDNS method 22 . Extracted RNA was stored at +4 °C during the preparation of the nested PCR if performed on the same day or −80 °C if delayed more than 24 h.…”

Section: Methodsmentioning

confidence: 99%

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study

Shaw,

Mampuela,

Lofiko

et al. 2023

Nat Microbiol

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Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6–30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory.

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Section: Methodsmentioning

confidence: 99%

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study

Shaw,

Mampuela,

Lofiko

et al. 2023

Nat Microbiol

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“…IC invested substantial effort in the research, development, and implementation of direct detection with nanopore sequencing (DDNS) for poliovirus surveillance [ 59 , 60 , 61 , 62 ]. This included publications that characterized rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing [ 59 ] and an analysis of the time taken to detect and respond to polio outbreaks in Africa considering the potential impact of direct molecular detection and nanopore sequencing [ 60 ].…”

Section: Resultsmentioning

confidence: 99%

“…This included publications that characterized rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing [ 59 ] and an analysis of the time taken to detect and respond to polio outbreaks in Africa considering the potential impact of direct molecular detection and nanopore sequencing [ 60 ]. IC also published a comparison of eleven RNA extraction methods for direct molecular detection of polioviruses from stool [ 61 ] and a prospective validation study of DDNS that reported preliminary cost estimates [ 62 ]. IC also included poliovirus surveillance in a consortium effort that it led with the goal of defining a research agenda for broad environmental wastewater surveillance of pathogens [ 63 ].…”

Section: Resultsmentioning

confidence: 99%

Review of Poliovirus Transmission and Economic Modeling to Support Global Polio Eradication: 2020–2024

Thompson,

Badizadegan

2024

Pathogens

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Continued investment in the development and application of mathematical models of poliovirus transmission, economics, and risks leads to their use in support of polio endgame strategy development and risk management policies. This study complements an earlier review covering the period 2000–2019 and discusses the evolution of studies published since 2020 by modeling groups supported by the Global Polio Eradication Initiative (GPEI) partners and others. We systematically review modeling papers published in English in peer-reviewed journals from 2020–2024.25 that focus on poliovirus transmission and health economic analyses. In spite of the long-anticipated end of poliovirus transmission and the GPEI sunset, which would lead to the end of its support for modeling, we find that the number of modeling groups supported by GPEI partners doubled and the rate of their publications increased. Modeling continued to play a role in supporting GPEI and national/regional policies, but changes in polio eradication governance, decentralized management and decision-making, and increased heterogeneity in modeling approaches and findings decreased the overall impact of modeling results. Meanwhile, the failure of the 2016 globally coordinated cessation of type 2 oral poliovirus vaccine use for preventive immunization and the introduction of new poliovirus vaccines and formulation, increased the complexity and uncertainty of poliovirus transmission and economic models and policy recommendations during this time.

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“…The optimized protocol for kit B was simpler and the reaction was faster than those of kit A; with the addition of all the primers at the start of reaction, the RT-PCR reaction finished within 2.5 h; however, the efficient amplification could negatively affect the detection rate of minor populations of PV strains in the mixtures ( Table 2 , see below discussion). Selection of commercially available kits is apparently a critical factor for the performance of the DD, including the extraction step of viral RNA [ 25 , 26 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Direct Detection Protocols for Poliovirus from Stool Samples of Acute Flaccid Paralysis Patients

Ueno,

Kitamura,

Nishimura

et al. 2023

Viruses

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Polio surveillance in the Global Polio Eradication Initiative has been conducted with virus isolation from stool samples of acute flaccid paralysis (AFP) cases. Under the current biorisk management/regulations, challenges arise in the timelines of the report, sensitivity of the test and containment of poliovirus (PV) isolates. In the present study, we evaluated protocols of previously reported direct detection (DD) methods targeting the VP1 or VP4–VP2 regions of the PV genome in terms of sensitivity and sequencability. An optimized protocol targeting the entire-capsid region for the VP1 sequencing showed a high sensitivity (limit of detection = 82 copies of PV genome) with a simpler and faster reaction than reported ones (i.e., with the addition of all the primers at the start of the reaction, the RT-PCR reaction finishes within 2.5 h). The DD methods targeting the VP1 region detected PV in 60 to 80% of PV-positive stool samples from AFP cases; however, minor populations of PV strains in the samples with virus mixtures were missed by the methods. Sequencability of the DD methods was primarily determined by the efficiency of the PCRs for both Sanger and nanopore sequencing. The DD method targeting the VP4–VP2 region showed higher sensitivity than that targeting the VP1 region (limit of detection = 25 copies of PV genome) and successfully detected PV from all the stool samples examined. These results suggest that DD methods are effective for the detection of PV and that further improvement of the sensitivity is essential to serve as an alternative to the current polio surveillance algorithm.

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Comparison of Eleven RNA Extraction Methods for Poliovirus Direct Molecular Detection in Stool Samples

Cited by 4 publications

References 25 publications

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study

Review of Poliovirus Transmission and Economic Modeling to Support Global Polio Eradication: 2020–2024

Evaluation of Direct Detection Protocols for Poliovirus from Stool Samples of Acute Flaccid Paralysis Patients

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