Discussion and knowledge
gaps
Sensitive molecular diagnostics through PCR and correct sampling and test interpretation are crucial for accurate COVID-19 diagnosis and thereby essential for good clinical practice. Furthermore, they are a key factor in outbreak phases where active case finding in combination with quarantine and contact tracing are crucial for outbreak control. We reviewed current knowledge on the kinetics of SARS-CoV-2 RNA shedding in different clinical samples of 32515 COVID-19 patients to inform clinical and public health decision making.
Our review comes with limitations like the choice of rapid instead of systematic style. Given the need for timely diagnostics information in the current pandemic and the overwhelming amount of publications indexed daily, we opted for a faster adapted systematic approach. We did not access the included publications for biases nor could fully trace for duplicate reporting of cases. Full comparability of the assays used could not be assured due to the lack of standardized diagnostic set-up across the world with respect to e.g. sampling materials, transport conditions, cell culture, extraction and PCR platforms. These were infrequently detailed in the publications. Furthermore, we did not review separately results from PCR assays targeting different parts of SARS-CoV-2 genome, for example E [10, 276, 277], RdRp [9, 276, 277], ORF1ab [11, 50, 51, 59, 276], ORF1a [276], ORF1b [276, 278], N [11, 50, 51, 59, 276, 278, 279] or S [9, 51, 59, 276, 280, 281] genes, though we tried to provide the authors’ estimations of viral loads or if not Ct values, where available. However, relatively few of the reviewed articles provided such detailed data, and we could not assess the data quality. We could not estimate to what extent the genetic drift in the SARS-CoV-2 genome affects the reported PCR results, nor could summarise false-positive or false-negative rates [282] due to insufficient data. Regular monitoring of the assays performance, detailed reporting and strict quality assurance mechanisms in accredited laboratories are vital to molecular diagnostics of SARS-CoV-2.
Respiratory tract specimens are the samples of choice for wide-spread screening and clinical course monitoring purposes, as well as for discharge and de-isolation [283, 284]. Further reports with well-documented sampling time points, comparing the different types of respiratory samples and their diagnostic window of use, are needed. Viral RNA concentrations in the URT peak in the early infection days, including in asymptomatic and mildly symptomatic cases. SARS-CoV-2 was successfully isolated from respiratory samples with data suggesting independent replication potential in both the upper and lower respiratory tract. Thus, respiratory sampling is the optimal strategy for both symptomatic and pre-/a-symptomatic cases and for the latter should be examined in conjunction with epidemiological evidence and clinical follow-up.
Although molecular detection on upper respiratory tract samples is the recommended method to diagnose a SARS-CoV-2 infection, serology is occasionally imperative to complement RT-PCR findings, e.g. in cases of insufficient clinical sensitivity of the RT-PCR over the disease course period [285]. In addition, serology can aid decision making on clinical and infection prevention management, e.g. by confirming the presence of SARS-CoV-2 specific (neutralizing) antibodies in cases with consistently low viral loads (high Ct values) in upper respiratory samples [286, 287]. SARS-CoV-2 RNA detection in the respiratory tract during convalescence can be prolonged. However, detection of viral genomes does not directly imply the presence of infectious virus and thereby infectivity. More information on the presence of viable SARS-CoV-2 infectivity is urgently needed, especially with de-isolation strategies in mind.
Although faecal-oral transmission has not been confirmed, it cannot be firmly excluded due to evidence of SARS-CoV-2 isolation from the GI tract [58, 271]. The detection of viable SARS-CoV-2 in faecal samples also has implications for the diagnostic possibilities of faeces tests. Laboratory diagnostic and safety protocols might need adjustments, for example by implementing inactivation steps and flow cabinet work. Stool and anal swabs are not samples of choice for screening purposes, though they might play a role in clinical monitoring. Some data suggested a shift from respiratory to GI shedding in the course of COVID-19 infection or independent replication in the GI tract. More information is needed on the significance of SARS-CoV-2 detection in faecal samples and how it relates to the timing of convalescence, as well as to the viral shedding specifics in children.
SARS-CoV-2 RNAemia has been detected mainly in severe cases and blood sampling could be useful in monitoring hospitalized patients. At this stage, there is no evidence to suggest SARS-CoV-2 could be transmitted through blood. More systematic data are needed to guide blood and organ transplantation safety protocols [288].
So far vertical transmission of SARS-CoV-2 seems unlikely or extremely rare with single reports on viral RNA detection in samples obtained during delivery. More studies on this route as well as potential transmission via sexual intercourse are needed. Viral RNA was detected in semen [235], but virus isolation was not attempted, and sexual transmission potential remains an open question. Data on SARS-CoV-2 kinetics in vulnerable groups like immunocompromised patients are insufficient. Neurological signs and syndromes associated with COVID-19 and the diagnostic/monitoring potential of CSF testing remain to be clarified.
In conclusion, using SARS-CoV-2 molecular testing by amplification techniques to the maximum of its potential is a combination of choosing the most appropriate sample type, collected with adequate sampling technique, and with the infection timeline in mind. Like all diagnostic techniques, it should be analysed considering strengths and limitations, and in conjunction with the clinical presentation and epidemiological evidence. Further well-documented systematic studies are needed to characterise fully the COVID-19 clinical course and guide public health decisions concerning optimal testing strategies.
Conflict of interest
None declared
Funding statement
KRS received fellowship funding by the European Public Health Microbiology Training Programme (EUPHEM), European Centre for Disease Prevention and Control (ECDC). An ECDC representative formally approved the manuscript before submission. MPGK received funding from the H2020 COVID-19 project RECOVER (grant agreement nr 101003589). The funders had no role in the collection, analysis, and interpretation of data, nor the writing of the review.
Acknowledgments
We would like to thank Nedyalko Vasilev for script development (used for data processing and visualization), and Jenny Deng for assisting with literature in Chinese. We also thank Silvia Herrera Leon and Kim Benschop for reviewing the manuscript. We thank GOARN-Research for the continuous partnership and helpful discussions on the work topics.
Authors’ contributions
CBEMR, LMK and KRS conceived the study design. KRS searched, compiled, and organised the information, and wrote the manuscript drafts. CBEMR, LMK, AAE, AM and MPGK reviewed the evidence and agreed on the conclusions. KRS and CBEMR finalised the manuscript text. All authors reviewed and contributed to the final manuscript.
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