aDetection only occurs if patients are followed up proactively from the time of exposure.
Ig, immunoglobulin; PCR, polymerase chain reaction; RT-PCR; real-time reverse transcription PCR; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2

Real-time reverse transcriptase polymerase chain reaction (rRT-PCR)

Real-time polymerase chain reaction (rRT-PCR) is the gold-standard molecular technique for the detection of SARS-CoV-2 viral RNA in all recommended samples. It targets the following sequences that code for structural viral proteins: spike (S), membrane (M), envelope (E), nucleocapsid (N), and RNA-dependent RNA polymerase (RdRP). Both S and N proteins are highly immunogenic (Ravi et al., 2020). The S proteins seems to be the major target of neutralizing antibodies for correlated coronaviruses (Berry et al., 2010). High infectivity of SARS-CoV-2 has compelled the CDC to publish rRT-PCR primers and probes together with all relevant literature for public access (Khalaf et al., 2020). The positive rate of rRT-PCR detection is dependent on the sample type, with differences between bronchoalveolar lavage fluid (93%), fibrobronchoscopy brush biopsy (46%), sputum (72%), nasal swabs (63%), pharyngeal swabs (32%), feces (29%), and blood (1%) (Wang et al., 2020). Combining nasopharyngeal and oropharyngeal swabs is now one of the most commonly used specimen types for diagnosing COVID-19 active infection (Lai and Lam, 2020). In September 2020, the WHO published a guideline not recommending saliva as the only specimen type for routine clinical diagnostics, because of the wide variation in collection methods (World Health Organization, 2020a).
The virus can be detected at least 48 hours before the onset of symptoms (pre-symptomatic cases), up to 12–14 days (at least 6–7 days) after the onset of symptoms in samples from the upper respiratory tract (NP/OP swabs), and for a median of 20 days in samples from the lower respiratory tract, including sputum, tracheal aspirate, and bronchoalveolar lavage (Mallett et al., 2020) (World Health Organization, 2020a) (Lippi et al., 2020).
Pooling rRT-PCR samples increases testing efficiency, which may be particularly helpful in areas with low prevalence and few health resources, given that only a limited number of tests are available (He et al., 2020). The idea is to pool samples from several individuals and test the combined sample with a single test. If the test is negative, all subjects are negative. If the test is positive, all individuals must be tested again to find the infected patient(s) (Food and Drug Administration USA, 2020). The US Food and Drug Administration initially proposed that five was the maximum number of samples to be pooled for rRT-PCR, but other studies found that the ideal number of pooled samples depends on the disease prevalence in the tested population (Hanel and Thurner, 2020) (Deckert et al., 2020) (Cherif et al., 2020). One potential constraint of pooled testing is that the false-negative rate may increase owing to dilution of positive samples, therefore high-sensitivity rRT-PCR tests are adequate to minimize this limitation (Lorentzen et al., 2020). In general, the larger the pool of specimens, the higher the likelihood of generating false-negative results (CDC, 2020a).
As with all diagnostic tests, the predictive value of rRT-PCR depends highly on its specificity, sensitivity, and prevalence of the disease in the target population (CDC, 2020a) (Table 1). False-negative results may also result from technical issues, from sampling to amplification, including thermal inactivation (Siam et al., 2020). A confirmatory test (e.g. repeated rRT-PCR) may be warranted if the initial results are negative, and the clinical characteristics are suggestive of infection (Lai and Lam, 2020) (Lorentzen et al., 2020) .