Automated sample-to-answer system for rapid and accurate diagnosis of emerging infectious diseases

Automated sample-to-answer systems that promptly diagnose emerging infectious diseases, such as zoonotic diseases, are crucial to preventing the spread of infectious diseases and future global pandemics. However, automated, rapid, and sensitive diagnostic testing without professionals and sample capacity and type limitations remains unmet needs. Here, we developed an automated sample-to-answer diagnostic system for rapid and accurate detection of emerging infectious diseases from clinical specimens. This integrated system consists of a microfluidic platform for sample preparation and a bio-optical sensor for nucleic acid (NA) amplification/detection. The microfluidic platform concentrates pathogens and NAs in a large sample volume using adipic acid dihydrazide and a low-cost disposable chip. The bio-optical sensor allows label-free, isothermal one-step NA amplification/detection using a ball-lensed optical fiber-based silicon micro-ring resonator sensor. The system is integrated with software to automate testing and perform analysis rapidly and simply;it can distinguish infection status within 80 min. The detection limit of the system (0.96 × 101 PFU) is 10 times more sensitive than conventional methods (0.96 × 102 PFU). Furthermore, we validated the clinical utility of this automated system in various clinical specimens from emerging infectious diseases, including 20 plasma samples for Q fever and 13 (11 nasopharyngeal swabs and 2 saliva) samples for COVID-19. The system showed 100% sensitivity and specificity for detecting 33 samples of emerging infectious diseases, such as Q fever, other febrile diseases, COVID-19, human coronavirus OC43, influenza A, and respiratory syncytial virus A. Therefore, we envision that this automated sample-to-answer diagnostic system will show high potential for diagnosing emerging infectious diseases in various clinical applications. © 2023 Elsevier B.V.

Multiplexed Ultrasensitive Sample-to-Answer RT-LAMP Chip for the Identification of SARS-CoV-2 and Influenza Viruses.

Prompt on-site diagnosis of SARS-CoV-2 with other respiratory infections would have minimized the global impact of the COVID-19 pandemic through rapid, effective management. However, no such multiplex point-of-care (POC) chip has satisfied a suitable sensitivity of gold-standard nucleic acid amplification tests (NAATs). Here, we present a rapid multiplexed ultrasensitive sample-to-answer LAMP (MUSAL) chip operated by simple LED-driven photothermal amplification to detect six targets from single-swab sampling. First, our MUSAL chip allows ultrafast on-chip sample preparation with ∼500-fold preconcentration at a rate of 1.2 mL min-1 . Second, our chip enables contamination-free amplification using autonomous target elution into on-chip reagents by photothermal activation. Finally, our chip accomplishes multiplexed on-chip diagnostics of SARS-CoV-2 and influenza viruses with a limit of detection (LoD) of 0.5 copies µL-1 . Our rapid, ultra-sensitive, cost-effective sample-to-answer chip with a multiplex capability will allow timely management of various pandemics situations we may face shortly. This article is protected by copyright. All rights reserved.

Automated sample-to-answer system for rapid and accurate diagnosis of emerging infectious diseases

Automated sample-to-answer systems that promptly diagnose emerging infectious diseases, such as zoonotic diseases, are crucial to preventing the spread of infectious diseases and future global pandemics. However, automated, rapid, and sensitive diagnostic testing without professionals and sample capacity and type limitations remains unmet needs. Here, we developed an automated sample-to-answer diagnostic system for rapid and accurate detection of emerging infectious diseases from clinical specimens. This integrated system consists of a microfluidic platform for sample preparation and a bio-optical sensor for nucleic acid (NA) amplification/detection. The microfluidic platform concentrates pathogens and NAs in a large sample volume using adipic acid dihydrazide and a low-cost disposable chip. The bio-optical sensor allows label-free, isothermal one-step NA amplification/detection using a ball-lensed optical fiber-based silicon micro-ring resonator sensor. The system is integrated with software to automate testing and perform analysis rapidly and simply;it can distinguish infection status within 80min. The detection limit of the system (0.96 × 101 PFU) is 10 times more sensitive than conventional methods (0.96 × 102 PFU). Furthermore, we validated the clinical utility of this automated system in various clinical specimens from emerging infectious diseases, including 20 plasma samples for Q fever and 13 (11 nasopharyngeal swabs and 2 saliva) samples for COVID-19. The system showed 100% sensitivity and specificity for detecting 33 samples of emerging infectious diseases, such as Q fever, other febrile diseases, COVID-19, human coronavirus OC43, influenza A, and respiratory syncytial virus A. Therefore, we envision that this automated sample-to-answer diagnostic system will show high potential for diagnosing emerging infectious diseases in various clinical applications.

Clinical Evaluation of BioFire COVID-19 Test, BioFire Respiratory Panel 2.1, and Cepheid Xpert Xpress SARS-CoV-2 Assays for Sample-to-Answer Detection of SARS-CoV-2.

Background: Due to the extreme infectivity of SARS-CoV-2, sample-to-answer SARS-CoV-2 reverse transcription (RT) polymerase chain reaction (PCR) assays are urgently needed in order to facilitate infectious disease surveillance and control. The purpose of this study was to evaluate three sample-to-answer SARS-CoV-2 RT-PCR assays­BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2­using clinical samples. Methods: A total of 77 leftover nasopharyngeal swab (NP) swabs (36 positives and 41 negatives) confirmed by reference SARS-CoV-2 RT real-time (q) PCR assay were collected. The clinical sample concordance, as specified by their respective emergency use authorizations (EUAs), in comparison to the reference SARS-CoV-2 RT-qPCR assay, was assessed. Results: The results showed that all three sample-to-answer SARS-CoV-2 RT-PCR assays provided perfectly concordant results consistent with the reference SARS-CoV-2 RT-qPCR assay. The BioFire COVID-19 Test exhibited the best turnaround time (TAT) compared to the other assays, regardless of the test results, using one-way analysis of variance followed by Scheffe's post hoc test (p < 0.001). The Xpert Xpress SARS-CoV-2 showed a shorter average TAT (mean ± standard deviation, 49.9 ± 3.1 min) in the positive samples compared to that (55.7 ± 2.5 min) of the negative samples. Conclusions: Our evaluation demonstrates that the BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2 assays compare favorably to the reference SARS-CoV-2 RT-qPCR assay, along with a 100% concordance in assay results for clinical samples and an acceptable analytical performance at their guaranteed limits of detection. The addition of a widely used simultaneous sample-to-answer SARS-CoV-2 RT-PCR assay will contribute to the number of medical laboratories able to test for COVID-19.

SAMPLE-TO-ANSWER WHOLE BLOOD SEROLOGICAL TEST FOR SARS-COV-2 USING THERMALLY RESPONSIVE VALVES

To maximize access to SARS-CoV-2 serological (antibody) tests, point-of-care (PoC) options are necessary. PoC tests require sample-to-answer functionality, which is challenging with whole blood. Here we demonstrate a sample-to-answer SARS-CoV-2 antibody test from whole blood using automated thermally actuated valves. Higher-order alkanes serve as partitions between immunoassay zones (sample/bind, rinse, detection);upon warming, the partitions are liquified, enabling magnetic beads to be pulled through each zone while continuing to partition the reagents. We demonstrate a detection limit of 0.7 ng/mL SARS-CoV-2 antibodies, multiple orders of magnitude lower than clinically relevant concentrations. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Evaluation of the Alinity m Resp-4-Plex Assay for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2, Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus.

The rapid emergence of the coronavirus disease 2019 (COVID-19) pandemic has introduced a new challenge in diagnosing and differentiating respiratory infections. Accurate diagnosis of respiratory infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is complicated by overlapping symptomology, and stepwise approaches to testing for each infection would lead to increased reagent usage and cost, as well as delays in clinical interventions. To avoid these issues, multiplex molecular assays have been developed to differentiate between respiratory viruses in a single test to meet clinical diagnostic needs. To evaluate the analytical performance of the FDA emergency use authorization (EUA)-approved Abbott Alinity m resp-4-plex assay (Alinity m) in testing for SARS-CoV-2, influenza A virus, influenza B virus, and respiratory syncytial virus (RSV), we compared its performance to those of both the EUA-approved Cepheid Xpert Xpress SARS-CoV-2, influenza A/B virus, and RSV assay (Xpert Xpress) and the EUA-approved Roche Cobas SARS-CoV-2 and influenza A/B virus assay (Cobas) in a single-center retrospective analysis. High concordance was observed among all three assays, with kappa statistics showing an almost perfect agreement (>0.90). The limit of detection (LOD) results for SARS-CoV-2 showed the Alinity m exhibiting the lowest LOD at 26 copies/mL, followed by the Cobas at 58 copies/mL and the Xpert Xpress at 83 copies/mL, with LOD results for the influenza A virus, influenza B virus, and RSV viral targets also showing equivalent or better performance on the Alinity m compared to the other two platforms. The Alinity m can be used as a high-volume testing platform for SARS-CoV-2, influenza A virus, influenza B virus, and RSV and exhibits analytical performance comparable to those of both the Xpert Xpress and Cobas assays. IMPORTANCE The rapid emergence of SARS-CoV-2 has introduced a new challenge in diagnosing and differentiating respiratory infections, especially considering the overlapping symptomology of many of these infections and differences in clinical interventions depending on the pathogen identified. To avoid these issues, multiplex molecular assays like the one described in this article need to be developed to differentiate between the most common respiratory pathogens in a single test and most effectively meet clinical diagnostic needs.

Clinical Comparison of Three Sample-to-Answer Systems for Detecting SARS-CoV-2 in B.1.1.7 Lineage Emergence.

PURPOSE: Accurate molecular diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, are needed for epidemiology studies and to support infection-control measures. We evaluated the analytical sensitivity and clinical performance of three sample-to-answer molecular-diagnostics systems for detecting SARS-CoV-2 using 325 nasopharyngeal swab clinical samples from symptomatic patients. METHODS: The BioFire Respiratory Panel 2.1 (RP2.1), cobas Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV platforms, which have been granted emergency-use authorization by the US FDA, were tested and compared. RESULTS: The positive percent agreement, negative percent agreement, and overall percent agreement among the three point of care testing systems were 98-100%, including for the wild-type SARS-CoV-2 (non-B.1.1.7) and a variant of concern (B.1.1.7). Notably, the BioFire RP2.1 may fail to detect the SARS-CoV-2 S gene in the B.1.1.7 lineage because of the spike protein mutation. CONCLUSION: All three point of care testing platforms provided highly sensitive, robust, and almost accurate results for rapidly detecting SARS-CoV-2. These automated molecular diagnostic assays can increase the effectiveness of control and prevention measures for infectious diseases.

A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing.

The outbreak of virus-induced infectious diseases poses a global public-health challenge. Nucleic acid amplification testing (NAAT) enables early detection of pandemic viruses and plays a vital role in preventing onward transmission. However, the requirement of skilled operators, expensive instrumentation, and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients. Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive, specific, and rapid viral nucleic acid testing. The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification (RT-LAMP) were integrated into the reaction units of a microfluidic disc. The whole processing steps such as injection of reagents, fluid actuation by rotation, heating and temperature control, and detection of fluorescence signals were carried out automatically by a customized instrument. We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) armored RNA particles. The estimated limit of detection for armored RNA particles is 2 copies per reaction, the throughput is 21 reactions per disc, and the assay sample-to-answer time is approximately 70 min. This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol, and can be readily adapted for virus detection outside the diagnostic laboratory. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s11426-020-9800-6 and is accessible for authorized users.

SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility.

BACKGROUND: Rapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. OBJECTIVES: We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert® Xpress SARS-CoV-2 and Mobidiag Novodiag® Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag® test in tertiary care emergency departments was assessed. RESULTS: In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert®, whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag®. Rapid SARS-CoV-2 testing with Novodiag® was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag® and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag® and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag®, but positive with the reference method with late Ct values. On average, a test result using Novodiag® was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. CONCLUSIONS: While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.