key: cord-0775904-yfw2uwkk authors: da Silva, Severino Jefferson Ribeiro; Pena, Lindomar J. title: A word of caution in interpreting COVID‐19 diagnostics tests date: 2020-09-28 journal: J Med Virol DOI: 10.1002/jmv.26531 sha: c26c1239b33d04f5993c948c6d0aaf5db88f9cd0 doc_id: 775904 cord_uid: yfw2uwkk nan To the Editor, Since the emergence of SARS-CoV-2, many diagnostic tests including molecular and serological assays have been developed and approved by the Food and Drug Administration (FDA) for diagnosis of COVID-19. 1, 2 However, concerns about the sensitivity and specificity of many diagnostic assays, especially the rapid tests, have been raised. Diagnostic tests with unacceptable rates of false positive and false-negative results interfere with therapeutic management of patients and can have serious implications for public health authorities in the decision-making process regarding COVID-19 control. In this context, understanding key concepts in terms of development and validation of diagnostic assays is crucial for correct interpretation of test results. The parameters for validating diagnostic tests include analytical sensitivity, analytical specificity, clinical sensitivity, clinical specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value (PPV), negative predictive value (NPV), repeatability, reproducibility, and accuracy (Table 1) . After initial development and optimization of a new COVID-19 test, the performance of the assay should be assessed using a set of welldefined clinical samples taking into account the required sample size. Ideally, data used for validation of the COVID-19 test should be published in a peer-reviewed publication to allow independent evaluation. Validation is essential for the development of a diagnostic test and requires a series of interrelated steps where the diagnostic test is experimentally standardized to detect the analyte (antibody, antigen, nucleic acid [DNA or RNA]), with precision and high accuracy. 3 Importantly, the new test should be compared side-by-side to a gold standard method that is used as a reference method to detect the pathogen. In the case of SARS-CoV-2, quantitative reverse tran- Analytical sensitivity/limit of detection Lowest concentration of the analyte that can be reliably detected by the assay. Analytical specificity Ability of the assay of not cross-reacting with other pathogens. Clinical sensitivity Probability the test is positive when the infection is present. Clinical specificity Probability the test is negative when the infection is absent. Positive likelihood ratio Ratio between the probability of an infected person testing positive and the probability of an uninfected person testing positive. Negative likelihood ratio Ratio between the probability of an infected person testing negative and the probability of an uninfected person testing negative. Positive predictive value (PPV) Probability that the pathogen is present when the test result is positive. Negative predictive value (NPV) Probability that the pathogen is absent when the test result is negative. Agreement between results of replicates of a sample both within and between runs of the same test in the same laboratory. Agreement between results of patient specimens assayed in different laboratories. Ability of the test to remain unaffected by minor variations that may occur during the testing process. Overall probability that the patient is correctly diagnosed by the test. symptoms (molecular or serological approach). Molecular assays should also consider the presence of mutations/mismatches in primer/probe binding sites in the SARS-CoV-2 genome that might interfere with viral detection and produce false-negative RT-qPCR results. 5 In the last few months, a variety of serological assays have been designed to detect antibodies against different portions of the SARS-CoV-2 genome. The viral nucleocapsid protein (N) and spike protein (S) have been the preferred antigens for use in serology because of their high antigenicity. 6 Recent studies suggested that the receptor-binding domain (RBD) of the viral spike protein (S1 subunit) is a major immunodominant epitope against which antibodies to SARS-CoV-2 are directed. The S1 subunit is more specific than S for the serological diagnosis of SARS-CoV-2 infection. 7, 8 Many studies have also reported false-negative results in serological tests. [9] [10] [11] In this context, Tang et al. 10 Clinical and laboratory diagnosis of SARS-CoV-2, the virus causing COVID-19 Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Serologic Testing Principles of validation of diagnostic assays for infectious diseases. Manual of Standards for Diagnostic Tests and Vaccines Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19 Sequence mismatch in PCR probes may mask the COVID-19 detection in Nepal Laboratory diagnosis of COVID-19: current issues and challenges The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients COVID-19: a meta-analysis of diagnostic test accuracy of commercial assays registered in Brazil Clinical performance of two SARS-CoV-2 serologic assays Diagnostic accuracy of serological tests for COVID-19: systematic review and metaanalysis