Reply.

To the Editor:

We welcome the opportunity to explain our findings further. First, we want to clarify that we included 6 expert microbiologists from the centers involved in the study. All co-authors played a fundamental role in the design and methodology of the study, as well as in the interpretation of the results. We want to especially highlight the input of microbiologists concerning these critical issues in our research.

Reverse transcriptase polymerase chain reaction (RT-PCR) positivity criteria are determined by the identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E and RdRp genes. However, there is currently no clear consensus on which cycle threshold marks the positivity of the RT-PCR test. The pandemic situation entails a shortage of microbiological diagnostic resources, which has not allowed Spanish microbiology laboratories to use a single RT-PCR technique. Furthermore, RNA extraction equipment also varies between laboratories. For all of these reasons, it would not be accurate to set a singular criterion for the required number of replicated genes and the cut-off values of cycle threshold. Regarding the reproducibility between laboratories, although the use of different techniques is a potential source of variability, all RT-PCR techniques used in the laboratories involved in this study are validated and accredited by the European Union. All subjects included were symptomatic (inclusion criteria) and have been reported qualitatively, therefore, in our opinion, the results are comparable.

At the time of the design of our study, we calculated the sample size choosing a prevalence of SARS-CoV-2 infection of 5% and an expected sensitivity of the antigen test of 90%. We collected data from the paired samples taken at participating hospitals after verbal consent and followed the Panbio Coronavirus Disease 2019 Ag Rapid Test Device (Abbott Rapid Diagnostic Jena GmbH) manufacturer's instructions and the implementation protocols of the sites. , As we describe in our report, patients with inclusion criteria were children age 0-16 years with symptoms compatible with SARS-CoV-2 infection within 5 days of attendance at an emergency department of 1 of 7 centers involved.

We agree that the validation of a diagnostic technique should be carried out with prospectively collected data. As mentioned in our discussion, this pilot study has allowed the working group (EPICO-AEP) to initiate a prospective validation study on the diagnostic accuracy of the Panbio SARS-CoV-2 antigen rapid test. We think that although the study has several limitations, already described, the information gained should be considered by clinicians and policymakers.