Résumé
RT-qPCR is used world-wide to test and trace the spread of SARS-CoV-2. Extraction-less or direct RT-PCR is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal (NP) or oral pharyngeal (OP) samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged ten global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international inter-laboratory ring trial. Participating labs were provided a common protocol, common reagents, aliquots of identical pooled clinical samples and purified nucleic acids, and used their existing in-house equipment. We observed 100% concordance across labs in the correct identification of all positive and negative samples, with highly similar Ct values observed. The test also performed well when applied to locally collected patient NP samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open access, direct RT-PCR assays are a feasible option for more efficient COVID-19 testing as demanded by the continuing pandemic.
Sujets)
COVID-19Résumé
Saliva has been described a less invasive and easy to handle sample, compared to nasopharyngeal swabs (NPS), in the diagnosis of COVID-19 in adults. Although the advantages of using saliva is still more evident in paediatric patients, little is now about its sensitivity in this group. The aim of this study was to compare the performance of saliva to that of NPS in the detection of SARS-CoV-2 in paediatric patients with mild symptoms. This study evaluated saliva samples from children with suspected COVID-19 who attended public healthcare services of Araraquara, Sao Paulo, Brazil. Children were asked to spit into a sterile container for collection of about 1ml of saliva after the NPS collection. SARS-COV-2 detection was performed by using the Altona RealStar(R) SARS-CoV-2 RT-PCR Kit 1.0. The sample consisted of 50 patients, in which 27 were girls (54%) and 23 were boys (46%). Ten were positive for SARS-CoV-2 in at least one sample collected. The mean age was 10.24 {+/-} 3.52 years old and saliva was collected after 4.76 {+/-} 1.31 days from the symptoms. Saliva and NPS have showed the same performance in the SARS-CoV-2 detection (k = 0.865, P < 0.001). In conclusion, saliva is a reliable alternative sample for COVID-19 diagnosis in paediatric population.
Sujets)
COVID-19Résumé
BackgroundThe detection of SARS-CoV-2 RNA by real-time polymerase chain reaction (PCR) in respiratory samples from COVID-19 patients is not a direct indication of the presence of viable viruses. The isolation of SARS-CoV-2 in cell culture system however, can acts as surrogate marker of infectiousness. Cell culture based studies performed mostly with hospitalized and moderate/severe COVID-19 claims that no replication competent virus is found after 9 days of the symptoms onset in respiratory samples. Therefore, it is now recommended 10 days isolation before patient discharge. MethodsWe cell-cultured 29 SARS-COV-2 RT-PCR positive respiratory samples at the 10th day after the illness in Vero E6 cells. After two passages, cytopathic effect and cycle threshold (CT) lower than the obtained in the original sample were used to determine positivity. FindingsWe found viable particles in (7/29) 24% of samples tested. The positivity in cell culture was strongly associated (p<0.0001) to the low cycle thresholds in clinical samples (Ct <21). ConclusionThis data adds important knowledge to the current protocols for de-isolation of patients with non-hospitalized mild COVID-19.