Dans une récente étude publiée sur medRxiv* serveur de préimpression, une équipe de chercheurs des États-Unis (US) a développé un flux de travail d’amplification isotherme médiée par une boucle de transcription inverse (RT-LAMP) basé sur la salive et sans extraction pour le coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2 ) détection.
La détection rapide de l’infection par le SRAS-CoV-2 suivie de l’isolement des sujets contagieux est la clé pour prévenir la transmission de la maladie à coronavirus 2019 (COVID-19). L’échantillonnage par écouvillonnage nasopharyngé pour la détection du COVID-19 est gênant pour les patients et nécessite des professionnels de la santé formés et des méthodes de diagnostic moléculaire laborieuses.
La salive est apparue comme une alternative efficace au prélèvement nasopharyngé pour les tests de surveillance du COVID-19 chez les personnes asymptomatiques présentant un taux d’excrétion virale élevé. Les méthodes RT-LAMP basées sur la salive sont explorées au milieu de la pandémie de COVID-19 parallèlement à la réaction en chaîne par polymérase quantitative par transcription inverse (RT-qPCR) car elle détecte rapidement les agents pathogènes avec une sensibilité et une spécificité élevées.
Il existe un besoin non satisfait d’une approche de test de diagnostic alternative et plus simple qui peut être largement mise en œuvre pour soutenir le système de santé débordé pendant la pandémie de COVID-19. Dans cette quête, les auteurs de cette étude ont développé un flux de travail de test rapide, simple et sensible basé sur la salive nécessitant des procédures d’échantillonnage minimales et un équipement sophistiqué.
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In this study, COVID-19 test results were collected, processed, and reported in compliance with Clinical Laboratory Improvement Amendments (CLIA). A total of 1,670 saliva samples were collected from 172 uninfected individuals between October 2021 to March 2021. The authors conducted a workplace surveillance program including 755 individuals that provide saliva samples one to three times per week between April 2021 to Feb 2022.
Optimization of the saliva sample preparation was performed for rapid release and viral ribonucleic acid (RNA) stabilization allowing reliable detection of SARS-CoV-2. The authors modified the various buffers to include pluronic F-68 surfactant referred to as saliva lysis buffer (SLB). Gamma-irradiated SARS-CoV-2 virions were used to spike saliva as a positive control.
In the RT-LAMP assay, the samples were each tested for SARS-CoV-2 (COVID LAMP) and actin (Actin LAMP). Finally, the time to reach the fluorescence detection threshold (Tt) was calculated.
Findings
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The findings of the study demonstrated that the sensitivity of RT-LAMP assay was increased on the addition of 0.68% pluronic F-68 surfactant. An optimal temperature of 95°C for 5 min for heating samples spiked with viral copies provided higher reproducibility across a range of viral loads.
The researchers assessed the association of RT-LAMP performance with different phases of saliva samples. They observed that SARS-CoV-2 was detected with the fastest amplification in saliva supernatant but not in saliva sediment or re-suspended sample.
Furthermore, the best performance of the RT-LAMP assay was observed with 1 µL of saliva samples. In saliva samples less than 1 µl, more variations were noted, and an increase in saliva volume delayed the reaction time of viral detection.
The robustness of the extraction-free RT-LAMP assay was measured from a large cohort of negative saliva samples spiked with 50 copies/µl of the SARS-CoV-2 virus. In the COVID-LAMP reaction, more than 98% of samples scored positive in colorimetric and fluorescent assay with 26 min cut-offs. Over 91% of samples scored COVID-positive in the fluorescent assay within 15 minutes. The authors observed a positive correlation between Tt values from all samples of COVID-19 and actin amplification reaction.
The authors demonstrated successful detection of a single SARS-CoV-2-positive specimen in a pool of randomly selected 16 negative saliva samples. Overall, the pooling strategy was beneficial when a specific saliva sample was colored, viscous, had low pH, and had interfering substances.
Extraction-free RT-LAMP assay demonstrated a sensitivity of 100%, 82%, and 63% when spiked with 40, 20, and 10 copies of viruses/µL, respectively, with a limit of detection (LOD) of 40 viral copies/µL of saliva.
Clinical validation studies using 30 pre-defined COVID-19-positive and COVID-19-negative saliva specimens showed 97% overall clinical sensitivity and 100% clinical specificity of RT-LAMP workflow demonstrating high accuracy of the assay.
The higher performance of RT-LAMP assay was observed compared to RT-qPCR when purified RNA was used as the input material. Only the samples with quantification cycle (Cq) values >34 in RT-qPCR failed to amplify in the RT-LAMP assay.
This workflow was implemented successfully in the CLIA laboratory setting for workplace surveillance and reporting. Interestingly, only 0.13% of samples generated inconclusive results highlighting the robustness of the test. The peaks in new cases followed a holiday period and mirrored the high infection rate in Massachusetts and nationwide.
Conclusion
The study demonstrated a robust and highly sensitive extraction-free, saliva-based RT-LAMP workflow with a simple colorimetric endpoint and fluorescence readout for SARS-CoV-2 detection. The assay had a rapid testing time and allowed quick tracing and isolation of COVID-19-positive subjects at the workplace to prevent further transmission.
The RT-LAMP assay enables an extensive and frequent SARS-CoV-2 diagnostic testing of the population with trace viral loads before symptom onset resulting in the effective management of the COVID-19 pandemic.
*Important notice
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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