Analytical and clinical performances of seven direct detection assays for SARS-CoV-2

Matsumura, Yasufumi; Yamazaki, Wataru; Noguchi, Taro; Yamamoto, Masaki; Nagao, Miki

doi:10.1016/j.jcvp.2023.100138

Cited by 4 publications

(6 citation statements)

References 20 publications

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“…In our previous study, the TRexGene assay showed 100% positive percent agreement and 100% negative percent agreement for both nasopharyngeal swabs and saliva samples when compared with the NIID N2 reference assay ( 17 ). Similarly, automation by the PCRpack system achieved 96.6% positive percent agreement and 100% negative percent agreement with the NIID N2 reference assay.…”

Section: Discussionmentioning

confidence: 89%

“…We employed TRexGene as a molecular assay because it can be performed without a nucleic acid purification step while showing high sensitivity and specificity, contributing to rapid and accurate testing ( 17 ). The defined LODs of the PCRpack system (1,000 copies/mL) were the same as the results from our previous study performed with a SARS-CoV-2 Detection Kit -Multi- (the former product name of TRexGene) by manual handling ( 17 ). This indicates that automation by the PCRpack system does not compromise analytical sensitivity.…”

Section: Discussionmentioning

confidence: 99%

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Development and evaluation of the automated multipurpose molecular testing system PCRpack for high-throughput SARS-CoV-2 testing

Matsumura,

Nakazaki,

Kitamori

et al. 2023

Microbiol Spectr

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Increasing the reliable testing capacity for SARS-CoV-2 is important for the diagnosis and control of COVID-19. We developed an automated, customizable, easy-to-implement molecular testing system, named PCRpack, for the high-throughput testing of SARS-CoV-2. PCRpack includes a liquid handling instrument enclosed in a negative-pressure clean booth (Biomek i5), a laboratory information management system (SimpPCR), other equipment, and documents that are needed for testing operation. An in vitro diagnostic assay was employed to detect SARS-CoV-2. System performance was evaluated based on liquid handling accuracy and precision, analytical sensitivity, clinical diagnostic performance, and testing capacity per day. Clinical diagnostic performance was determined against the reference extraction-based reverse transcription-PCR assay using 3,965 upper respiratory samples. Analytical sensitivity analysis showed a lower limit of detection of 1,000 genome copies/mL of sample. The accuracy and precision of sample or reagent dispensing in PCRpack ranged from −2.24% to 0.73% and 0.83% to 4.52%, respectively. In the evaluation of clinical samples, PCRpack showed a positive percent agreement of 96.6% and a negative percent agreement of 100% compared with the reference assay. The average turnaround times per 94 samples and the maximum numbers of tests within an 8-hour shift of one operator were 2 hours and 28 minutes vs 2 hours and 1 minute and 564 vs 376 samples for PCRpack and the manual method, respectively. The developed PCRpack system shows high liquid handling and clinical diagnostic performance and is a promising testing system for increasing the SARS-CoV-2 testing capacity and testing future emerging pathogens. IMPORTANCE Accurate and fast molecular testing is important for the diagnosis and control of COVID-19. During patient surges in the COVID-19 pandemic, laboratories were challenged by a higher demand for molecular testing under skilled staff shortages. We developed an automated multipurpose molecular testing system, named PCRpack, for the rapid, high-throughput testing of infectious pathogens, including SARS-CoV-2. The system is provided in an all-in-one package, including a liquid handling instrument, a laboratory information management system, and other materials needed for testing operation; is highly customizable; and is easily implemented. PCRpack showed robust liquid handling performance, high clinical diagnostic performance, a shorter turn-around time with minimal hands-on time, and a high testing capacity. These features contribute to the rapid implementation of the high-performance and high-throughput molecular testing environment at any phase of the pandemic caused by SARS-CoV-2 or future emerging pathogens.

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Development and evaluation of the automated multipurpose molecular testing system PCRpack for high-throughput SARS-CoV-2 testing

Matsumura,

Nakazaki,

Kitamori

et al. 2023

Microbiol Spectr

Self Cite

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“…The advantages of pooling molecular testing over rapid antigen tests include high sensitivity and the ability to detect pathogens for which rapid antigen tests are unavailable (e.g., at the early stage of the COVID-19 pandemic and future emerging pathogens). Our previous evaluation revealed that the LOD of the Fujirebio rapid antigen test was >1,000 fold greater than that of the three developed pooling systems ( 22 ). A retrospective study that analyzed >1 million subjects estimated the sensitivity of the Veritor rapid antigen test for asymptomatic individuals to be 38.5%‒84.2% compared with that of the RT-PCR assay ( 27 ).…”

Section: Discussionmentioning

confidence: 98%

“…We previously investigated the analytical sensitivities of 11 commercial or in-house RT‒PCR assays (based on nonautomated, manual methodology) with the same ATCC heat-inactivated strain used in this study and found that six assays achieved LODs of <500 copies/mL, followed by two assays with LODs of <1,000 copies/mL ( 21 ). Among four commercial direct RT‒PCR assays that bypass the nucleic acid purification step, two assays showed LODs of 1,000–2,500, and the others showed LODs of >10,000 copies/mL ( 22 ). Based on these findings, we predefined the minimum LOD levels for individual and pooled testing of the developed automated systems as 1,000 and 2,500 copies/mL, respectively, to achieve similar levels of performance to manual RT-PCR assays or direct RT-PCR assays.…”

Section: Discussionmentioning

confidence: 99%

Development and evaluation of three automated media pooling and molecular diagnostic systems for the detection of SARS-CoV-2

Matsumura,

Noguchi,

Shinohara

et al. 2024

Microbiol Spectr

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Pooled testing combined with molecular diagnostics for the detection of SARS-CoV-2 is a promising method that can increase testing capacities and save costs. However, pooled testing is also associated with the risks of decreased test sensitivity and specificity. To perform reliable pooled testing, we developed and validated three automated media pooling and molecular diagnostic systems. These pooling systems (geneLEAD-PS, Panther-PS, and Biomek-PS) comprised existing automated molecular detection platforms, corresponding automated media pooling devices, and laboratory information management systems. Analytical sensitivity analysis and mock sample evaluation were performed, and the obtained data were used to determine the sizes of the pool for the validation study. In the validation study, a total of 2,448, 3,228, and 6,420 upper respiratory samples were used for geneLEAD-PS, Panther-PS, and Biomek-PS, respectively, and the diagnostic performances were compared with the reference RT‒PCR assay. A pool size of 6 for geneLEAD-PS and a pool size of 4 for Panther-PS and Biomek-PS were selected for the validation studies. All three systems showed high positive percent agreement values of ≥90.5% and negative percent agreement values of ≥99.8% for any specimen type. Pooled testing resulted in a 65%–71% reduction in cost per sample. The testing capacities of geneLEAD-PS, Panther-PS, and Biomek-PS were 144 samples in 3 hours, 384 samples in 5.5 hours, and 376 samples in 4 hours, respectively. The developed pooling systems showed robust diagnostic performances and will increase the testing capacities of molecular diagnostic tests while saving costs and may contribute to infection control of COVID-19. IMPORTANCE During the COVID-19 pandemic, there have been surges in demand for accurate molecular diagnostic testing and laboratory supply shortages. Pooled testing combined with highly sensitive molecular testing, which entails mixing multiple samples as a single sample, is a promising approach to increase testing capacities while reducing the use of consumables. However, pooled testing is associated with risks that compromise diagnostic performance, such as false negatives due to dilution of positive samples or false positives due to cross-contamination. To perform reliable pooled testing, three different pooling systems (an automated pooling device, an automated molecular detection platform, and a laboratory information management system) were developed to accurately interpret pooled testing results. These three systems were validated using multiple clinical samples and showed high concordance with individual testing. The developed pooling systems will contribute to increasing reliable molecular testing capacities while using fewer consumables and saving costs.

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“…However, the implementation of real-time RT-PCR requires specialized laboratories, infrastructure and personnel, and costly reagents and equipment, which are often not widely available in LMICs. Antigen detection based rapid diagnostic tests (Ag-RDTs) represent an attractive alternative, because of their low cost and ease of operation, but concerns about their sensitivity limit their use at particularly low viral loads [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

Laboratory Evaluation of a SARS-CoV-2 RT-LAMP Test

2023

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There is a need to have more accessible molecular diagnostic tests for the diagnosis of severe acute respiratory syndrome coronavirus 2 disease in low- and middle-income countries. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) may provide an attractive option as this technology does not require a complex infrastructure. In this study, the diagnostic performance of a SARS-CoV-2 RT-LAMP was evaluated using RT-PCR-confirmed clinical specimens of COVID-19-positive (n = 55) and -negative patients (n = 55) from the Netherlands. The observed sensitivity of the RT-LAMP test was 97.2% (95% CI: 82.4–98.0%) and the specificity was 100% (95% CI: 93.5–100%). The positive predictive value of the RT-LAMP was 100%, the negative predictive value 93.2% (95% CI: 84.3–97.3%), and the diagnostic accuracy was 96.4% (95% CI: 91.0–99.0%). The agreement between the RT-LAMP and the RT-PCR was “almost perfect” (κ-value: 0.92). The evaluated RT-LAMP might provide an attractive alternative molecular diagnostic tool for SARS-CoV-2 in resource limited settings.

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Analytical and clinical performances of seven direct detection assays for SARS-CoV-2

Cited by 4 publications

References 20 publications

Development and evaluation of the automated multipurpose molecular testing system PCRpack for high-throughput SARS-CoV-2 testing

Development and evaluation of the automated multipurpose molecular testing system PCRpack for high-throughput SARS-CoV-2 testing

Development and evaluation of three automated media pooling and molecular diagnostic systems for the detection of SARS-CoV-2

Laboratory Evaluation of a SARS-CoV-2 RT-LAMP Test

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