key: cord-0769993-kma08sxi
authors: Scotta, Marcelo Comerlato; de David, Caroline Nespolo; Varela, Fernanda Hammes; Sartor, Ivaine Tais Sauthier; Polese-Bonatto, Márcia; Fernandes, Ingrid Rodrigues; Zavaglia, Gabriela Oliveira; Ferreira, Charles Francisco; Kern, Luciane Beatriz; Santos, Amanda Paz; Krauzer, João Ronaldo Mafalda; Pitrez, Paulo Márcio; de Almeida, Walquiria Aparecida Ferreira; Porto, Victor Bertollo Gomes; Stein, Renato T.
title: Low performance of a SARS-CoV-2 point-of-care lateral flow immunoassay in symptomatic children during the pandemic()
date: 2021-06-03
journal: J Pediatr (Rio J)
DOI: 10.1016/j.jped.2021.04.010
sha: 2d9a0d469f5766157ea3a2556e2751b91f37e43e
doc_id: 769993
cord_uid: kma08sxi

OBJECTIVE: To evaluate the accuracy of an antibody point-of-care lateral flow immunoassay (LFI - Wondfo Biotech Co., Guangzhou, China) in a pediatric population. METHODS: Children and adolescents (2 months to 18 years) with signs and symptoms suggestive of acute SARS-CoV-2 infection were prospectively investigated with nasopharyngeal RT-PCR and LFI at the emergency room. RT-PCR was performed at baseline, and LFI at the same time or scheduled for those with less than 7 days of the clinical picture. Overall accuracy, sensitivity and specificity were assessed, as well as according to the onset of symptoms (7-13 or ≥14 days) at the time of LFI test. RESULTS: In 175 children included, RT-PCR and LFI were positive in 51 (29.14%) and 36 (20.57%), respectively. The overall sensitivity, specificity, positive and negative predictive value was of 70.6% (95%CI 56.2-82.5), 96.8% (95%CI 91.9-99.1), 90.0% (95%CI 77.2-96.0), and 88.9% (95%CI 83.9-92.5), respectively. At 7-13 and ≥14 days after the onset of symptoms, sensitivity was 60.0% (95%CI 26.2-87.8) and 73.2% (95%CI 57.1-85.8) and specificity was 97.9% (95%CI 88.7-99.9) and 96.1% (95%CI 89.0-99.2), respectively. CONCLUSION: Despite its high specificity, in the present study, the sensitivity of LFI in children was lower (around 70%) than most reports in adults. In acute care settings, although a positive result is informative, a negative LFI test cannot rule out COVID-19 in children.

The COVID-19 pandemic is the largest world health crisis of the century. [1, 2] The limitations of the effectiveness of the containment measures and the great impact in several aspects of society forced world leaders to make difficult decisions. [3] [4] [5] The most evidencebased strategies to mitigate the pandemic included the widespread use of accurate testing. [6] Despite the large number of studies published since the beginning of the pandemic, the knowledge of SARS-CoV-2 infection test accuracy is still evolving. As critical cases of COVID-19 were far more common in older adults and patients with comorbidities, most studies assessing the accuracy of diagnostic tests were performed in this population. [7] [8] [9] [10] Although children and adolescents with COVID-19 usually have a mild disease, severe cases occur, [11] as is the case of the rare COVID-19-related multisystem inflammatory syndrome. This syndrome resembles Kawasaki disease and can lead to multiorgan failure and even death. [12] [13] [14] Also, children may play a significant role in SARS-CoV-2 spread, mimicking what happens with other respiratory viruses. Although differences in viral load in the pediatric population are still controversial, some studies report that infected children shed SARS-CoV-2 virus with nasopharyngeal viral loads comparable to, or even higher than in adults. [15] [16] [17] All this novel information justifies more precise definitions of the best accuracy of viral diagnostic tools, as an important public health preventive measure among the pediatric population.

The aim of this study was to assess the accuracy of an antibody LFI test in children and adolescents compared to RT-PCR in the acute care setting. The authors have also compared the test accuracy between infants and older children.

This is a prospective multicenter observational study with data collected in two hospitals, from May to November 2020. Pediatric patients (> 2 months and < 18 years) admitted at emergency rooms (ERs) or visiting outpatient clinics with signs or symptoms suggestive of COVID-19 (cough, fever, or sore throat) within 14 days of onset of symptoms were eligible. SARS-CoV-2 antibodies bind to antigen-dye conjugate and to colloidal gold complexes, defining a line test (T) in the kit's window, presenting as a dark-colored band. Instead, samples without SARS-CoV-2 specific antibodies will not display this dark-colored band, indicating a negative result. All valid tests presented a positive control band (C). If this control band was not visible, the test was considered invalid and the participant retested. If there was doubt about the result another researcher evaluated the result or the test was repeated.

RT-PCR sample collection was performed at baseline and LFI at the same time or scheduled for later in those with less than 7 days of clinical onset. All tests were performed through a standardized protocol by the trained study team. The laboratory team was blind to the LFI test results until the final analysis. However, when LFI was not performed at the inclusion, the research team had access to the RT-PCR test results. To explore the accuracy according to the onset of symptoms, the authors calculated days of symptoms onset at the time of LFI testing, and assessed sensitivity and specificity at 7-13 days, and ≥ 14 days. The authors performed a sensitivity sub-analysis to compare the accuracy of LFI between infants (< 2 years old) and older children (≥ 2 years old). Patients without both LFI and RT-PCR results were excluded from the analysis. All analyses were performed in R version 3.6.3.

383 subjects were screened for the study, and 208 excluded: 16 for not meeting inclusion criteria, 13 for not consenting, and 179 did not perform LFI, as shown in Figure 1 Table 3 ).

The analysis to assess the test accuracy between infants (< 2 years old) and older children (≥ 2 years old) did not show significant differences. LFI test overall sensitivity and specificity for those < 2 years old (n = 36, PCR+ =10) were 70% (95%CI 34.8-93.3) and 100% (95%CI 86.8-100.0) respectively, while for children ≥ 2 years old (n = 139, PCR+ = 41) sensitivity was 70.7% (95%CI 54.5-83.9) and specificity 95.9% (95%CI 89.9-98.9).

To the authors' knowledge, this is the first study testing LFI accuracy in the pediatric population in a prospective study during the COVID-19 pandemic. The specificity found was over 96% regardless of days after the onset of symptoms. However, the sensitivity of LFI was low (73.2%), even lower during the second week after disease onset. This finding is in contrast with the commercial test description and illustrates the importance of clinical studies to validate any test in a specific population. Accuracy did not differ between infants and older children.

The authors found a lower sensitivity than in most other reports, but it is important to point out that the performance of these tests may change according to different manufacturers. Previous studies in adults have shown sensitivity numbers around 80%-90%. [18] [19] [20] [21] The exact immunologic specificities between SARS-CoV-2 infected children and adults are still unclear and under investigation. Possible explanations may include a lower expression of angiotensin-converting enzyme 2 receptors, immature humoral and cellular immune responses, and different innate and adaptive responses in children when compared to adults. [22] [23] [24] The rapid point-of-care diagnosis was identified as a research priority by WHO R&D Blue Print expert group.

[25] Mean days of seroconversion for adult COVID-19 confirmed cases is around 9-12 days post-onset. [25, 26] Some studies describe almost 100% of seroconversion in adult patients within 3 weeks of symptoms onset. [27] Although there are no previous reports of the accuracy of serologic LFI in children and adolescents, studies about serology in this age group may be a proxy for the understanding of both tests.

Serological diagnosis is especially important for patients who seek health care late for a molecular test.

The present study's results suggest that in acute care settings a positive result may confirm the diagnosis, but a negative result is not sufficient to rule out the disease in children.

If used in epidemiological surveys, the limitations in sensitivity must be considered for the interpretation of such data. It may be useful to evaluate clusters of cases, since a COVID-19 early diagnosis may allow confirmation of subsequent cases by epidemiological criteria.

Despite these limitations, LFI testing is simple to perform and has a low cost, with potential use especially in low-resource or remote settings. This study has some limitations. Firstly, 179 patients did not return for LFI testing.

Pediatric patients with COVID-19 usually have a good prognosis and caregivers probably had the concern to return to a hospital setting for further testing when the clinical evolution was mild. Patients who did not return were younger, but the test performance was not different between age groups. A higher proportion of caucasian children did not undergo LFI testing, however, the authors believe that such differences would not change the study's overall results. Still, individuals who did not return could have a milder clinical course. Although controversial, mild disease has been related to lower levels of antibodies. [28] Therefore, due to this subject loss in the study, the sensitivity found could be overestimated. Also, RT-PCR was used as the reference diagnostic method even with limitations such as the timing for collection and sensitivity around 71%, [29] [30] which might have led to an underestimation in specificity and overestimation in sensitivity observed for the LFI test. Finally, in the present sample, COVID-19 prevalence was around 30%. Interpretation of these results must be careful in different epidemiological scenarios, as accuracy (especially PPV and NPV) may change according to prevalence.

Despite the high specificity (> 96%) of LFI in a pediatric population, the sensitivity was lower than reports among adults. Further studies are necessary to assess the long-term performance of this test in children, as it is not known how long antibodies remain positive after an acute infection.

The authors declare no conflicts of interest. 

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The authors thank the Scientific Committee of the Research Support Nucleus (NAP) of