Более чувствительный метод определения вируса показал (ретроспективно), что поле для исследований среди педиатричсеких пациентов - гораздо шире, чем предполагалось ранее.
Whereas reverse transcription and subsequent quantitative PCR (RT-qPCR) are key methods for the302detection of SARS-CoV-2 and local as well as global pandemic surveillance, it is well known that several confounding factors can lead to false-negative results. It seems clear that the time course of 304SARS-CoV-2 load in the days after infection massively influences the predictive value of the RT- qPCR tests. A recent study discovered a median false-negative rate as high as 38% (CI, 18% to 65%) even at the day of symptom onset, although concomitantly SARS-CoV-2 load seems to be close to its highest levels. Moreover, few days before and after symptom onset the false-negative SARS-CoV-2 discovery rate by RT-qPCR seems to worsen dramatically . Besides the time point of specimens sampling post-infection, heterogeneities in specimens sampling technique, transportation, storage conditions, nucleic acids purification, laboratory equipment, staff experience but also RT-qPCR conditions might be among the most important factors influencing test qualities.
Therefore, we aimed to develop a confirmatory test for a stably expressedSARS-CoV-2 sgRNA 335amplicon target that can complement RT-qPCR strategies without disturbing established and automatable laboratory workflows. In particular, we intended to develop a pyrosequencing assay that would allow, subsequent to RT-qPCR, the categorical confirmation of SARS-CoV-2 infections in acute cases, where the clinical suspicion is high, but the SARS-CoV-2 infection cannot be ruled out by 9RT-qPCR alone.The proposed pyrosequencing approach does not negatively affect preceding RT-340qPCR pipelines in SARS-CoV-2 diagnostics and can therefore add important value to RT-qPCR, where this method alone delivers conflicting results. Particularly, this can happen close to the detection limits qPCR, practically CTvalues >30
Exemplarily for the implementation of the developed experimentalpipelinefor anepidemiological 368survey, we conducted a field studyin search for SARS-CoV-2 infected patientswithoutobvious 369commonSARS-CoV-2associated symptoms, since the majority of cases pediatric SARS-CoV-2 infectionsdevelop only very mild disease coursesю
In a very recent UK national cohort study on neonatal SARS-CoV-2 infection 66 SARS-CoV-2-positive babies could be identified, who at day of presentation exhibitedhyperthermia, poor feeding, vomiting, coryza, other respiratory signs and lethargy as the most common signs of infection or, respectively, no signs of infection at all .On 374the other hand,an unknown fraction of SARS-CoV-2 infectionsin childhood seem tofundamentally differ from adults and can be more heterogeneous in their presentation. The most striking example is Multisystem Inflammatory Syndrome in Children (MIS-C), a rare SARS-CoV-2-induced Kawasaki- like hyperinflammatory syndrome[5, 6]. Another recent study reports that children and adults can exhibit a very different antibody responses upon SARS-CoV-2-infections across the clinical spectrum of associated diseases, which do not obligatorily match the adult COVID-19 spectrum . Necessarily, the association unusual symptoms with acute infections will contribute to our understanding about the heterogeneity of SARS-CoV-2-borne diseases in general and particularly in children. Using twolarge pediatric cohorts for a field study, we have successfully demonstrated in this study thatcombined RT-qPCR/pyrosequencing is a reliable tool that allows the faithful confirmation 384of less distinctive or asymptomatic cases with SARS-CoV-2 infection close to the detection limits of RT-qPCR.