Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System.

BACKGROUND: The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study. METHODS: A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader. RESULTS: The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL. CONCLUSION: The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays.

SARS-CoV-2 Detection via RT-PCR in Matched Saliva and Nasopharyngeal Samples Reveals High Concordance in Different Commercial Assays.

BACKGROUND: Self-collected saliva samples can increase the diagnostic efficiency and benefit healthcare workers, patient care, and infection control. This study evaluated the performance of self-collected saliva samples compared to nasopharyngeal swabs using three commercial kits for the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Matched nasopharyngeal and saliva samples were collected from 103 patients with either asymptomatic or symptomatic COVID-19. Both samples were evaluated using three commercial kits (TaqCheck, Allplex, and TaqPath). To evaluate sample stability, viral RNA extraction was performed in the presence or absence of an RNA-stabilizing solution. Storage conditions, including the duration, temperature, and stability after freezing and thawing of the samples, were also evaluated. RESULTS: All the saliva samples showed 100% concordance with the nasopharyngeal swab results using TaqCheck and Allplex kits, and 93% using TaqPath kit. No difference was observed in the samples that used the RNA-stabilizing solution compared to the group without the solution. The Ct values of the freeze-thawed samples after 30 days were higher than those on day 0; however, the results were consistent the fresh samples. CONCLUSION: The high concordance of SARS-CoV-2 detection via reverse transcription-polymerase chain reaction (RT-PCR) in matched saliva and nasopharyngeal samples using different commercial assays reinforces the concept that self-collected saliva samples are non-invasive, rapid, and reliable for diagnosing SARS-CoV-2 infection.

SARS-CoV-2 variant of concern P.1 (Gamma) infection in young and middle-aged patients admitted to the intensive care units of a single hospital in Salvador, Northeast Brazil, February 2021.

OBJECTIVES: To evaluate changes in the characteristics of patients with coronavirus disease 2019 (COVID-19) after the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) P.1 (Gamma), by comparing the clinical, demographic, and laboratory profiles of patients hospitalized during the first (May to July 2020) and second (December 2020 to February 2021) pandemic waves. METHODS: Data were collected from the records of COVID-19 patients (n = 4164) admitted to a single hospital in Salvador, Northeast Brazil. SARS-CoV-2 genome sequencing was performed on nasopharyngeal swab samples from 12 patients aged <60 years admitted to the intensive care unit (ICU) in February 2021. RESULTS: Between June 2020 and February 2021, the median age of patients admitted to the ICU decreased from 66 to 58 years (P < 0.05). This was accompanied by an increased proportion of patients without comorbidities (15.32% vs 32.20%, P < 0.0001). A significant reduction in the cycle threshold values of SARS-CoV-2 RT-PCR tests was observed in the second wave (P < 0.0001). Sequencing analysis detected lineage Gamma in all 12 ICU patients sampled in February 2021. CONCLUSIONS: The results of this study demonstrated an increased proportion of younger adults without comorbidities with severe disease during the second COVID-19 wave, shortly after the confirmation of local Gamma circulation.

Early detection of P.1 variant of SARS-CoV-2 in a cluster of cases in Salvador, Brazil.

We report 3 cases of severe COVID-19 due to the SARS-CoV-2 P.1 lineage in a familial cluster detected in Salvador, Bahia-Brazil. All cases were linked to travel by family members from the state of Amazonas to Bahia in late December 2020. This report indicates the cryptic transmission of the SARS-CoV-2 P.1 lineage across Brazil and highlights the importance of genomic surveillance to track the emergence of new SARS-CoV-2 variants of concern.

Genomic Evidence of SARS-CoV-2 Reinfection Involving E484K Spike Mutation, Brazil.

Uncertainty remains about how long the protective immune responses against severe acute respiratory syndrome coronavirus 2 persists, and suspected reinfection in recovered patients has been reported. We describe a case of reinfection from distinct virus lineages in Brazil harboring the E484K mutation, a variant associated with escape from neutralizing antibodies.