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Front Cell Infect Microbiol ; 11: 653616, 2021.
Article in English | MEDLINE | ID: covidwho-1315950


Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4-100.0%) and 98.6% (95% CI: 94.9-99.8%), respectively, showing high consistency (Cohen's kappa, 0.986; 95% CI: 0.966-1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

COVID-19 , SARS-CoV-2 , COVID-19 Testing , Clinical Laboratory Techniques , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Reverse Transcription , Sensitivity and Specificity
Rev Peru Med Exp Salud Publica ; 38(1): 7-16, 2021.
Article in Spanish, English | MEDLINE | ID: covidwho-1289342


OBJECTIVES: To standardize and validate an in-house RT-LAMP test for the detection of SARS-CoV-2, based on laboratory and field assays using samples from COVID-19 suspected patients. MATERIALS AND METHODS: An in-house SARS-CoV-2 RT-LAMP molecular test was standardized, establishing the detection limit with Vero cells of isolated Peruvian strains of SARS-CoV-2, and the robustness to various concentrations of primers. The laboratory validation was performed with 384 nasal and pharyngeal swab samples (UFH) obtained between March and July 2020. The field validation was performed with 383 UFH obtained from COVID-19 suspected symptomatic cases. All samples were tested by RT-LAMP and RT-qPCR. The RT-qPCR was considered as the reference standard test. The concordance measures and diagnostic performance were calculated. RESULTS: The detection limit was consistent in cases with Ct <30 in both tests, showing efficiency to detect up to 1000 copies/µL of the target gene. Robustness was evidenced with half of the primer concentrations and 20 µL of final volume. Absence of amplification was identified for other HCoVs. Concordance showed a kappa index of 0.88 (95% CI: 0.83-0.93) and 0.89 (95% CI: 0.84 - 0.94) in laboratory and field settings, respectively. The sensitivity value in the laboratory was 87.4% (95% CI: 80.8 - 92.4) and 88.1% in the field (95% CI: 81.6 - 92.9). The specificity value in both settings was 98.8% (95% CI: 96.4-99.7). CONCLUSIONS: The in-house SARS-CoV-2 RT-LAMP test was successfully validated based on its adequate robustness, no cross-reactions, good concordance, and diagnostic performance compared to RT-qPCR.

OBJETIVOS: Estandarizar una prueba RT-LAMP in house para la detección de SARS-CoV-2 y validarla con muestras de laboratorio y de campo en pacientes con sospecha clínica de COVID-19. MATERIALES Y MÉTODOS: Se estandarizó una prueba molecular RT-LAMP in house para la detección de SARS-CoV-2 estableciéndose el límite de detección con células Vero de cepas peruanas aisladas de SARS-CoV-2. Se validó la prueba en laboratorio con 384 muestras de hisopado nasal y faríngeo (HNF) obtenidas entre marzo y julio de 2020. Para la validación de campo se obtuvieron muestras de HNF de 383 casos sintomáticos sospechosos de COVID-19. Todas las muestras fueron evaluadas por RT-LAMP y RT-qPCR. Para la validación de laboratorio y de campo se consideró como estándar de referencia al RT-qPCR, se calcularon medidas de concordancia y rendimiento diagnóstico. RESULTADOS: El límite de detección fue consistente en los casos con umbral de ciclo (Ct) Ct < 30 en ambas pruebas, mostrando eficiencia para detectar hasta 1000 copias/µL del gen diana. Se evidenció robustez con la mitad de las concentraciones de cebadores y 20 µL de volumen final. Se identificó ausencia de amplificación para otros coronavirus humanos. La concordancia en laboratorio obtuvo un Kappa de 0,88 (IC 95%: 0,83-0,93) y en campo fue de 0,89 (IC 95%: 0,84−0,94); la sensibilidad en laboratorio fue de 87,4% (IC 95%: 80,8−92,4) y en campo fue de 88,1% (IC 95%: 81,6−92,9), la especificidad en ambos escenarios fue de 98,8% (IC 95%: 96,4−99,7). CONCLUSIONES: La prueba RT-LAMP in house fue validada por presentar una adecuada robustez, sin reacciones cruzadas, buena concordancia y rendimiento diagnóstico comparado con el RT-qPCR.

COVID-19 , SARS-CoV-2 , Animals , Chlorocebus aethiops , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral , Reference Standards , Sensitivity and Specificity , Vero Cells