key: cord-0813516-drw791c5 authors: Pérez-García, Felipe; Pérez-Tanoira, Ramón; Romanyk, Juan; Arroyo, Teresa; Gómez-Herruz, Peña; Cuadros-González, Juan title: Alltest rapid lateral flow immunoassays is reliable in diagnosing SARS-CoV-2 infection from 14 days after symptom onset: a prospective single-center study date: 2020-05-27 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104473 sha: 776758ca493152ca04df004984b8cdd0cdd549ec doc_id: 813516 cord_uid: drw791c5 OBJECTIVES: SARS-CoV-2 infection diagnosis is challenging in patients from 2-3 weeks after the onset of symptoms, due to the low positivity rate of the PCR. Serologic tests could be complementary to PCR in these situations. The aim of our study was to analyze the diagnostic performance of one serologic rapid test in COVID-19 patients. METHODS: We evaluated a lateral flow immunoassay (AllTest COVID-19 IgG/IgM) which detects IgG and IgM antibodies. We validated the serologic test using serum samples from 100 negative patients (group 1) and 90 patients with COVID-19 confirmed by PCR (group 2). Then, we prospectively evaluated the test in 61 patients with clinical diagnosis of pneumonia of unknown etiology that were negative for SARS-CoV-2 by PCR (group 3). RESULTS: All 100 patients from group 1 were negative for the serologic test (specificity = 100%). Regarding group 2 (PCR-positive), the median time from their symptom onset until testing was 17 days. For these 90 group-2 patients, the test was positive for either IgM or IgG in 58 (overall sensitivity = 64.4%), and in patients tested 14 days or more after the onset of symptoms, the sensitivity was 88.0%. Regarding the 61 group-3 patients, median time after symptom onset was also 17 days, and the test was positive in 54 (88.5% positivity). CONCLUSIONS: Our study shows that serologic lateral flow immunoassays are reliable as a complement of PCR to diagnose SARS-CoV-2 infection after 14 days from the onset of symptoms and in patients with pneumonia and negative PCR for SARS-CoV-2. The pandemic due to SARS-CoV-2 that started in Wuhan in December 2019 (1, 2) has caused until May 16, 2020, a total of 4,425,485 cases and 302,059 deaths worldwide (3) . Spain is the country of the European region that has been most affected by the infection, accounting for 230,183 cases and 33,998 deaths by May 16 (3) . From the beginning of the pandemic, one of the main concerns was the complexity and excessive time to results of the diagnostic test, based on polymerase chain reaction (PCR) (4, 5) . Few clinical microbiology laboratories were prepared at this time to process such a massive volume of samples that grew exponentially. In our hospital, which is a medium-sized center (490 beds), from March 5 to April 6, a total of 7,453 respiratory samples (the vast majority nasopharyngeal exudates) were processed for SARS-CoV-2 PCR, reaching a positivity rate between 20 and 40%. Another problem was the low positivity rate of nasopharyngeal samples in patients presenting a clinical syndrome compatible with COVID-19 in the second and third week of infection (1, (6) (7) (8) , which is generally the period in which patients are admitted to the hospital (1) . Besides, most patients presented a non-productive cough (9) , and this fact, together with the high risk of generating aerosols in bronchoscopies explains that most respiratory samples came from the upper respiratory tract, where the virus concentration is lower beyond the first week after the onset of symptoms (8, 10) . As a consequence, the positivity rate of the PCR in these patients could be lower than expected and many of them were hospitalized with a provisional diagnosis of pneumonia of unknown etiology and possible COVID- These limitations have led to development of different serologic microplate ELISA tests (11, 12) . Recently published studies confirm the usefulness of combining PCR in nasopharyngeal exudates with the detection of IgM and IgG antibodies in the J o u r n a l P r e -p r o o f blood (13) . The combination of molecular and serologic techniques allowed some authors to achieve a sensitivity of 97% for diagnosis of SARS-CoV-2 infection (11) . However, those time-consuming tests based on ELISA are not as suitable for clinical use as rapid tests and, as a matter of fact, cannot be included in the management algorithms in emergency departments (11, (13) (14) (15) . Since the beginning of the epidemic in Spain, information emerged about the availability of rapid serological diagnostic kits that detected IgG and IgM antibodies using immunochromatographic (ICT) tests. However, there are very few published studies about the clinical application of these kits (15) . Our aim was to evaluate the diagnostic performance of one of these serologic rapid tests, first by a validation of the test in negative control patients and confirmed cases of COVID-19, and then by a prospective evaluation in patients with pneumonia of unknown etiology and a clinical diagnosis of COVID-19 with negative PCR for SARS-CoV-2. We included three groups of patients in our study: Demographic and clinical variables of the study population were obtained from the medical records (age, sex, hospital and ICU admission, outcome and disease J o u r n a l P r e -p r o o f severity). Severity of infection was classified according to WHO criteria. Briefly, patient infections were classified as: mild disease, pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis and septic shock (16) . The time from the onset of symptoms was calculated in groups 2 and 3 from the day of onset of symptoms to the day of the extraction of the serum sample. The serologic test was evaluated on clinical samples from groups 1 and 2 in order to assess the sensitivity and specificity of the test: Group 1 (negative controls): they were used to evaluate the specificity of the serological test. 100 aliquots of cryopreserved sera, corresponding to 100 different controls, were recovered from the serum archive. Group 2: (patients with positive PCR for SARS-CoV-2): they were used to evaluate the sensitivity of the serological test, using PCR as a gold standard. A total of 90 confirmed cases of SARS-CoV-2 infection were included, and cryopreserved aliquots of serum of those patients were used. Those aliquots were previously obtained from samples sent to the laboratory to carry out other serologies. The assessment was performed on patients from group 3 (pneumonia of unknown etiology with negative PCR for SARS-CoV-2). Fresh serum samples from these 61 patients were studied. We considered a positive result for samples in which IgG, IgM or both of them were detected. Continuous variables were expressed as median and interquartile range (IQR) and categorical variables as proportions. Comparisons between continuous variables were made using the t test or Mann-Whitney test, depending on the J o u r n a l P r e -p r o o f normality of the distribution. For these comparisons, a p value less than or equal to 0.05 was considered significant. Specificity and sensitivity were calculated for the serologic tests using the results from group 1 and group 2 patients, respectively. Statistical analysis was performed using Stata/IC 13.1 (StataCorp, Texas, USA). A total of 251 patients were studied. Median age was 61 years (IQR: 46-74) and 152 (60.6%) were males. The overall serologic results from the three groups of patients are summarized in Table 1 J o u r n a l P r e -p r o o f We assessed the serologic test in the group 3 patients (patients with pneumonia of unknown etiology and negative PCR). There were no patients with less than 7 days Our study shows that Alltest COVID-19 IgG/IgM lateral flow immunoassay is a reliable tool to diagnose SARS-CoV-2 infection from 14 days of onset of symptoms, being especially useful in hospitalized patients with pneumonia of unknown etiology with 14 or more days from the onset of symptoms and in whom the PCR has been negative. The current situation of the COVID-19 pandemic requires an urgent and coordinated answer to the inherent problems of the PCR-based diagnosis: on the one hand the low capacity to carry out PCR techniques in some laboratories and also the low sensitivity of PCR test in nasopharyngeal samples, specially from the second week of infection (2, 6). This study shows that the AllTest COVID-19 IgG/IgM rapid test for the detection of IgG and IgM is very specific (100%) and reaches a sensitivity of 88.0% from day 14 of onset of symptoms in patients with previous positive PCR in a nasopharyngeal exudate. According to our data, the vast majority of patients seroconvert from day 14 and this is a key aspect in the management of health care personnel (17) and in population immunity studies related to pandemic control (18) . There is increasing evidence on the usefulness of serology for diagnosis of SARS-CoV-2 infection, but most of these studies were based on microplate ELISA tests to detect IgA, IgM and IgG antibodies (11) (12) (13) . These techniques have shown high sensitivity and specificity, but they also require special equipment, trained personnel and take several hours to perform. Due to this, there is an increasing interest about the usefulness of serologic rapid tests, but there is scarce information about their diagnostic performance. In a recently published study, Li et al. (15) performed a multicenter evaluation of a serologic rapid test that the authors had developed. In their study, the overall sensitivity was 88.7% and the specificity was 90.6%. However, these authors did not present any data about the time after the J o u r n a l P r e -p r o o f onset of symptoms except from 58 out of 525 patients enrolled in the study. Moreover, for this subgroup of patients they only described that the time from the onset of symptoms was 8 to 33 days. As this is a serological test, this kind of information is key in order to interpret properly the sensitivity and specificity commercial LFAs, but their usefulness is questionable due to a lack of official performance validations (21) . Because of this, it is recommended to perform a validation of each diagnostic test before its clinical implementation and consequently, our results should not be extrapolated to other available commercial LFAs. The question about the reliability of serologic rapid tests is still under debate (22, 23) and more research is needed on this topic. We think that our study may help to point out the usefulness of this rapid test. Alltest COVID-19 IgG/IgM lateral flow immunoassay is reliable to diagnose SARS-CoV-2 infection as a complement of PCR from 14 days after the onset of symptoms. This test was especially useful in hospitalized patients with pneumonia and negative PCR for SARS-CoV-2. 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Science. 2020. 22. World Health Organization. Advice on the use of point-of-care immunodiagnostic tests for COVID-19 Performance of VivaDiag COVID-19 IgM/IgG Rapid Test is inadequate for diagnosis of COVID-19 in acute patients referring to emergency room department We want to thank Carolyn Brimley Norris, from the University of Helsinki Language Services for her help with the preparation of the manuscript. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.