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In the Global Polio Laboratory Network (GPLN), poliovirus (PV) screening results from acute flaccid paralysis (AFP) surveillance is based on virus isolation (VI) through cell culture, entailing long turnaround times and the amplification of live poliovirus. An alternative Direct Detection strategy (DD-ITD) for screening viral nucleic acid from stools, bypassing the need for virus culture, has been developed and extensively validated by GPLN partners. A multi-laboratory demonstration project was conceived to field-test the DD-ITD method by GPLN laboratories from the WHO African, Western Pacific, and Eastern Mediterranean regions, where wild serotype 1 or vaccine-derived polioviruses still circulate. Strategically selected laboratories were tasked to simultaneously process stool suspensions with the current gold-standard VI method and the new DD-ITD strategy. Results from 12 laboratories were compiled and analyzed to assess the quality of each RNA extraction and rRT-PCR run. Matched results for both methods of over 10,500 specimens showed an overall method agreement of 91%. All laboratories detected more PV presumptive positive samples with the DD-ITD strategy than with VI, but a large proportion of DD-ITD positive results (72%) were inconclusive or non-typeable, requiring confirmation through sequencing. A total of 298 (2.8%) samples were PV positive using both methods, 828 (7.9%) positive only for DD-ITD, and 62 (0.6%) positive only with VI. The DD-ITD overall performance, quality of results, and agreement between method results varied significantly across participating laboratories. DD-ITD implementation would entail building proficiency in advanced molecular laboratory techniques and data analysis, and increased demand for confirmatory sequencing. IMPORTANCE Surveillance of acute flaccid paralysis (AFP) and sensitive poliovirus detection are key components of the WHO Global Polio Eradication Strategy. This work summarizes the results of a multi-laboratory evaluation designed to field-test the performance and applicability of a molecular Direct Detection strategy (DD-ITD) that does not require amplification of live poliovirus. AFP samples were processed in parallel with both the DD-ITD and the current gold-standard PV detection methodology, based on virus isolation (VI) through cell culture. All participating laboratories detected more PV presumptive positive samples using the DD-ITD strategy than with virus isolation methodology, although a higher proportion of DD-ITD results required confirmatory sequencing. Significant variability among laboratories was observed in the quality of the results and overall DD-ITD performance. Implementing DD-ITD would entail building proficiency in advanced molecular laboratory techniques and strengthening data analysis skills.
In the Global Polio Laboratory Network (GPLN), poliovirus (PV) screening results from acute flaccid paralysis (AFP) surveillance is based on virus isolation (VI) through cell culture, entailing long turnaround times and the amplification of live poliovirus. An alternative Direct Detection strategy (DD-ITD) for screening viral nucleic acid from stools, bypassing the need for virus culture, has been developed and extensively validated by GPLN partners. A multi-laboratory demonstration project was conceived to field-test the DD-ITD method by GPLN laboratories from the WHO African, Western Pacific, and Eastern Mediterranean regions, where wild serotype 1 or vaccine-derived polioviruses still circulate. Strategically selected laboratories were tasked to simultaneously process stool suspensions with the current gold-standard VI method and the new DD-ITD strategy. Results from 12 laboratories were compiled and analyzed to assess the quality of each RNA extraction and rRT-PCR run. Matched results for both methods of over 10,500 specimens showed an overall method agreement of 91%. All laboratories detected more PV presumptive positive samples with the DD-ITD strategy than with VI, but a large proportion of DD-ITD positive results (72%) were inconclusive or non-typeable, requiring confirmation through sequencing. A total of 298 (2.8%) samples were PV positive using both methods, 828 (7.9%) positive only for DD-ITD, and 62 (0.6%) positive only with VI. The DD-ITD overall performance, quality of results, and agreement between method results varied significantly across participating laboratories. DD-ITD implementation would entail building proficiency in advanced molecular laboratory techniques and data analysis, and increased demand for confirmatory sequencing. IMPORTANCE Surveillance of acute flaccid paralysis (AFP) and sensitive poliovirus detection are key components of the WHO Global Polio Eradication Strategy. This work summarizes the results of a multi-laboratory evaluation designed to field-test the performance and applicability of a molecular Direct Detection strategy (DD-ITD) that does not require amplification of live poliovirus. AFP samples were processed in parallel with both the DD-ITD and the current gold-standard PV detection methodology, based on virus isolation (VI) through cell culture. All participating laboratories detected more PV presumptive positive samples using the DD-ITD strategy than with virus isolation methodology, although a higher proportion of DD-ITD results required confirmatory sequencing. Significant variability among laboratories was observed in the quality of the results and overall DD-ITD performance. Implementing DD-ITD would entail building proficiency in advanced molecular laboratory techniques and strengthening data analysis skills.
In the Global Poliovirus Laboratory Network (GPLN), participation and successful completion in annual proficiency test (PT) panels has been a part of the WHO accreditation process for decades. The PT panel is a molecular external quality assessment (mEQA) that evaluates laboratory preparedness, technical proficiency, the accuracy of data interpretation, and result reporting. Using the Intratypic Differentiation (ITD) real-time RT-PCR kits from CDC, laboratories run screening assays and report results in accordance with the ITD algorithm to identify and type polioviruses. The mEQA panels consisted of 10 blinded, non-infectious lyophilized RNA transcripts, including programmatically relevant viruses and targets contained in the real-time PCR assays. Sample identities included wildtype, vaccine-derived (VDPV), Sabin-like polioviruses, enterovirus, and negatives, as well as categories of invalid and indeterminate. The performance of individual laboratories was assessed based on the laboratory’s ability to correctly detect and characterize the serotype/genotype identities of each sample. The scoring scheme assessed the laboratory readiness following GPLN guidelines. Laboratories receiving mEQA scores of 90 or higher passed the assessment, scores of less than 90 failed and required remedial actions and re-evaluation. In 2021 and 2022, 123 and 129 GPLN laboratories were invited to request the annual PT panel, and 118 and 127 laboratories submitted results, respectively. The overall results were good, with 86% and 91.5% of laboratories passing the PT panel on their first attempt in 2021 and 2022, respectively. Most labs scored the highest score of 100, and less than one quarter scored between 90 and 95. Less than 10% of submitting laboratories failed the PT, resulting in in-depth troubleshooting to identify root causes and remediations. Most of these laboratories were issued a second PT panel for repeat testing, and almost all laboratories passed the repeat PT panel. The results of the 2021 and 2022 annual mEQA PTs showed that, despite the COVID-19 pandemic, the performance remained high in the GPLN, with most labs achieving the highest score. For these labs, the real-time PCR assay updates that were implemented during 2021–2022 were carried out with full adherence to procedures and algorithms. Even initially failing labs achieved passing scores after remediation.
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