Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis
Fecha
2024
Tipo
artículo original
Autores
Segura Ulate, Ismael
Apú Leitón, Navilla
Cortés Ledezma, Bernal
Zaldívar, Yamitzel
Querol Audi, Jordi
Ortega, Carlos Alexander
Flores Mora, Fernando Esteban
Madrigal Redondo, German Leonardo
Gatica Arias, Andrés Mauricio
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Background: the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other. Methods: we tested alternative diagnostic methods using saliva, heatinduced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP. Results: RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88-96% sensitivity and 95-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction. Discussion: our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. Nonetheless, use of direct saliva after a HIRR and detected with RT-LAMP does not produce an acceptable diagnostic performance.
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SALIVA SAMPLE, ALTERNATIVE PROTOCOL, COVID-19