key: cord-0853017-czfw63yu authors: Mallett, S.; Allen, J.; Graziadio, S.; Taylor, S. A.; Sakai, N. S.; Green, K.; Suklan, J.; Hyde, C.; Shinkins, B.; Zhelev, Z.; Peters, J.; Turner, P.; Roberts, N. W.; Ferrante di Ruffano, L.; Wolff, R.; Whiting, P.; Winter, A.; Bhatnagar, G.; Nicholson, B. D.; Halligan, S. title: At what times during infection is SARS-CoV-2 detectable and no longer detectable using RT-PCR based tests?: A systematic review of individual participant data date: 2020-07-14 journal: nan DOI: 10.1101/2020.07.13.20152793 sha: 915f18b1a9a86fed8c5fea43c631ab3e0f90121a doc_id: 853017 cord_uid: czfw63yu Background Tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral ribonucleic acid (RNA), using reverse transcription polymerase chain reaction (RT-PCR) are pivotal to detecting current coronavirus disease (COVID-19) and duration of detectable virus indicating potential for infectivity. Methods We conducted an individual participant data (IPD) systematic review of longitudinal studies of RT-PCR test results in symptomatic SARS-CoV-2. We searched PubMed, LitCOVID, medRxiv and COVID-19 Living Evidence databases. We assessed risk of bias using a QUADAS-2 adaptation. Outcomes were the percentage of positive test results by time and the duration of detectable virus, by anatomical sampling sites. Findings Of 5078 studies screened, we included 32 studies with 1023 SARS-CoV-2 infected participants and 1619 test results, from -6 to 66 days post-symptom onset and hospitalisation. The highest percentage virus detection was from nasopharyngeal sampling between 0 to 4 days post-symptom onset at 89% (95% confidence interval (CI) 83 to 93) dropping to 54% (95% CI 47 to 61) after 10 to 14 days. On average, duration of detectable virus was longer with lower respiratory tract (LRT) sampling than upper respiratory tract (URT). Duration of faecal and respiratory tract virus detection varied greatly within individual participants. In some participants, virus was still detectable at 46 days post-symptom onset. Interpretation RT-PCR misses detection of people with SARS-CoV-2 infection; early sampling minimises false negative diagnoses. Beyond ten days post-symptom onset, lower RT or faecal testing may be preferred sampling sites. The included studies are open to substantial risk of bias so the positivity rates are probably overestimated. Tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral ribonucleic acid (RNA), using reverse transcription polymerase chain reaction (RT-PCR) are pivotal to detecting current coronavirus disease and duration of detectable virus indicating potential for infectivity. We conducted an individual participant data (IPD) systematic review of longitudinal studies of RT-PCR test results in symptomatic SARS-CoV-2. We searched PubMed, LitCOVID, medRxiv and COVID-19 Living Evidence databases. We assessed risk of bias using a QUADAS-2 adaptation. Outcomes were the percentage of positive test results by time and the duration of detectable virus, by anatomical sampling sites. Of 5078 studies screened, we included 32 studies with 1023 SARS-CoV-2 infected participants and 1619 test results, from -6 to 66 days post-symptom onset and hospitalisation. The highest percentage virus detection was from nasopharyngeal sampling between 0 to 4 days post-symptom onset at 89% (95% confidence interval (CI) 83 to 93) dropping to 54% (95% CI 47 to 61) after 10 to 14 days. On average, duration of detectable virus was longer with lower respiratory tract (LRT) sampling than upper respiratory tract (URT). Duration of faecal and respiratory tract virus detection varied greatly within individual participants. In some participants, virus was still detectable at 46 days postsymptom onset. RT-PCR misses detection of people with SARS-CoV-2 infection; early sampling minimises false negative diagnoses. Beyond ten days post-symptom onset, lower RT or faecal testing may be preferred sampling sites. The included studies are open to substantial risk of bias so the positivity rates are probably overestimated. There are numerous reports of negative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) test results in participants with known SARS-CoV-2 infection, and increasing awareness that the ability of RT-PCR tests to detect virus depends on the timing of sample retrieval and anatomical sampling site. Individual studies suggest that positive test results from RT-PCR with nasopharyngeal sampling declines within a week of symptoms and that a positive test later in the disease course is more likely from sputum, bronchoalveolar lavage (BAL) or stool, but data are inconsistent. We searched 5078 titles and abstracts for longitudinal studies reporting individual participant data (IPD) for RT-PCR for participants with COVID-19 linked to either time since symptom onset or time since hospitalisation. Search included SARS-CoV-2 and RT-PCR keywords and MeSH terms. Each included study was subject to careful assessment of risk of bias. This IPD systematic review (SR) addresses RT-PCR test detection rates at different times since symptom onset and hospitalisation for different sampling sites, and summarises the duration of detectable virus. To our knowledge, this is the first rapid SR addressing this topic. We identified 32 studies available as published articles or pre-prints between January 1 st and April 24 th 2020, including participants sampled at 11 different sampling sites and some participants sampled at more than one site. At earlier time points, nasopharyngeal sampling had the highest virus detection, but the duration of shedding was shorter compared to lower respiratory tract sampling. At 10 to 14 days post-symptom onset, the percentage of positive nasopharyngeal test results was 54% compared to 89% at day 0 to 4. Presence and duration of faecal detection varied by participant, and in nearly half duration was shorter than respiratory sample detection. Virus detection varies for participants and can continue to be detected up to 46 days post-symptom onset or hospitalisation. The included studies were open to substantial risk of bias, so the detection rates are probably overestimates. There was also poor reporting of sampling methods and sparse data on . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07. 13.20152793 doi: medRxiv preprint sampling methods that are becoming more widely implemented, such as self-sampling and short nasal swab sampling (anterior nares/mid turbinate). Results from this IPD SR of SARS-CoV-2 testing at different time points and using different anatomical sample sites are important to inform strategies of testing. For prevention of ongoing transmission of SARS-CoV-2, samples for RT-PCR testing need to be taken as soon as possible post-symptom onset, as we confirm that RT-PCR misses more people with infection if sampling is delayed. The percentage of positive RT-PCR tests is also highly dependent on the anatomical site sampled in infected people. Sampling at more than one anatomical site may be advisable as there is variation between individuals in the sites that are infected, as well as the timing of SARS-CoV-2 virus detection at an anatomical site. Testing ten days after symptom onset will lead to a higher frequency of negative tests, particularly if using only upper respiratory tract sampling. However, our estimates may considerably understate the frequency of negative RT-PCR results in people with SARS-CoV-2 infection. Further investment in this IPD approach is recommended as the amount data available was small given the scale of the pandemic and the importance of the question. More studies, learning from our observations about risk of bias and strengths of example studies (Box 1, Box 2) are urgently needed to inform the optimal sampling strategy by including self-collected samples such as saliva and short nasal swabs. Better reporting of anatomical sampling sites with a detailed methodology on sample collection is also urgently needed. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07.13.20152793 doi: medRxiv preprint Accurate testing is pivotal to controlling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), otherwise known as the coronavirus disease 2019 (COVID-19). Considerable political and medical emphasis has been placed on rapid access to testing both to identify infected individuals so as to direct appropriate therapy, appropriate return to work, and to implement containment measures to limit the spread of disease. However, success depends heavily on test accuracy. Understanding when in the disease course the virus is detectable is important for two purposes, firstly to understand when and how to detect SARS-CoV-2, and secondly to understand how long individuals are likely to remain infective posing a risk to others. The success of COVID-19 testing depends heavily on the use of accurate tests at the appropriate time. Testing for active virus infection relies predominantly on reverse transcription polymerase chain reaction (RT-PCR), which detects viral ribonucleic acid (RNA) that is shed in varying amounts from different anatomical sites and at different times during the disease course. It is increasingly understood that differences in virus load impact directly on diagnostic accuracy, notably giving rise to negative tests in disease-positive individuals 1 . Positivity is contingent upon sufficient virus being present to trigger a positive test which may depend on test site, sampling methods and timing 2 . For example, it is believed that positive nasopharyngeal RT-PCR declines within a week of symptoms so that a positive test later in the disease course is more likely from sputum, bronchoalveolar lavage fluid or stool 3 . Nomenclature for anatomical site is also unclear, with a wide variety of overlapping terms used such as "oral", "throat", "nasal", "pharyngeal", "nasopharyngeal". Because testing is pivotal to management and containment of COVID-19, we performed an individual participant data (IPD) systematic review of emerging evidence about test accuracy by anatomical sampling site to inform optimal sampling strategies for SARS-CoV-2. We aimed to examine at what time points during SARS-CoV-2 infection it is detectable at different anatomical sites using RT-PCR based tests. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07.13.20152793 doi: medRxiv preprint This IPD systematic review followed the recommendations of the PRISMA-IPD checklist 4 . Eligible articles were any case-series or longitudinal studies reporting participants with confirmed COVID-19 tested at multiple times during their infection and provided IPD for RT-PCR test results at these times. We stipulated that test timings were linked to index dates of time since symptom onset or time since hospital admission as well as COVID-19 diagnosis by positive RT-PCR and/or suggestive clinical criteria, for example World Health Organization (WHO) guidelines 5 . Search strings were designed and conducted subsequently in PubMed, LitCOVID and medRxiv by an experienced information specialist (NR). The search end date was 24 th April 2020. We additionally included references identified by COVID-19: National Institute for Health Research (NIHR) living map of living evidence (http://eppi.ioe.ac.uk/COVID19_MAP/covid_map_v4.html), COVID-19 Living Evidence (https://ispmbern.github.io/covid-19/living-review/) with a volunteer citizen science team, "The Virus Bashers". Additional details S1. Data were extracted into pre-specified forms. We did not contact authors for additional information. Study, participant characteristics, and ROB were extracted in Microsoft Excel We could not identify an ideal risk-of-bias (ROB) tool for longitudinal studies of diagnostic tests, so we adapted the risk of bias tool for diagnostic accuracy studies QUADAS-2 6 to . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07.13.20152793 doi: medRxiv preprint include additional signalling questions to cover anticipated issues. ROB signalling questions, evaluation criteria and domain assessment of potential bias are reported (S2). Details of sampling sites and methods, including location of the sampling site(s), any sample grouping (for example, if combined throat and nasal swabs), were extracted from full texts by a clinician (NS) with queries referred to a second clinician (ST). If stated, details of sampling methodology were recorded, including who collected samples, information regarding anatomical location (e.g. how the nasopharynx was identified), and sample storage. Additional details S3. IPD RT-PCR results were extracted from each article and converted to binary results ("positive" or "negative"). Data from Kaplan-Meier (KM) curves were extracted using Web digitizer 7 . Additional details S3. Days since symptom onset and days since hospital admission were calculated from reported IPD. Data were presented collated across 5-day time intervals for each sample method, with longer times grouped within the longest time interval, and 95% CI were calculated for proportions. For comparison of duration of positive RT-PCR from respiratory tract (RT) and faecal samples, analysis and graphical presentation was restricted to participants sampled by both methods. Data analysis used STATA (14.2 StataCorp LP, Texas, USA). Additional details S3. Funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07.13.20152793 doi: medRxiv preprint 5078 articles were identified, 116 full text articles were screened, and 32 articles were included (Figure 1 ). Most articles were from China, in hospitalised adults participants (Table 1) . Articles reported on a total of 1023 participants and 1619 test results. Twenty-six (81%) articles reported data on test results since the start of symptoms, 23 (72%) since hospital admission. Sixteen studies including 22% (229/1023) of the participants reported both these time points: The median time between symptom onset and hospitalisation was 5 days (interquartile range (IQR) 2 to 7 days). The median number of participants per study was 22 (IQR 9 to 56, range 5 to 232) and the median number of RT-PCR test results per participant was 4 (IQR 2 to 9) ( Table 2) . Articles variably specified sampling sites according to anatomical location, or grouped more than one site for analysis, for example as upper RT (see S4). The most frequent sample sites were faeces (n=13), nasopharyngeal (n=10) and throat (n=9), although there was a range of other sites including blood, urine, semen, and conjunctival swabs ( Table 2 ). Details of sampling method were generally absent. Two studies specified the person taking the samples. One study described how the nasopharynx was identified and the swab technique The sampling sites yielding the greatest proportion of positive tests were nasopharyngeal, throat, sputum, or faeces. Insufficient data were available to evaluate saliva and semen. Only 33% of participants who were tested with blood samples had detectable virus . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. We further grouped sites into upper (URT) and lower (LRT) respiratory tract. The rate of sample positivity reduced faster from URT sites compared to LRT sites ( Figure 4A ). Given that analysis across all participants is likely to be influenced by preferential URT sampling of participants with less severe disease, we also analysed participants who underwent both URT and LRT sampling. Again, URT sites on average cleared faster (median 12 days, 95% CI 8 to 15 days) than LRT sites (median 28 days, 95% CI 20 to not estimable; Figure 4B ); the majority of participants clear virus from URT site before LRT ( Figure 4C ). Data based on time since hospital admission are consistent with data for time since symptom onset. Across participants sampled by both RT and faecal sampling since hospital admission, 29% of participants were detected using RT sampling but not by faecal sampling, (52/177 participants, 95% CI 23 to 37%, 10 studies). The time to RT-PCR tests becoming undetectable varied greatly by participant, although time to undetectable virus was similar for both sampling sites ( Figure 5 ), in participants with RT-PCR test results from both RT and faecal . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . samples. Thirty nine out of 89 participants (44%, 95% CI 33 to 55%) had a shorter duration of detection in faecal samples than in RT samples. Median time to clearance from RT was shorter in participants based on time since hospitalisation (125 participants, p=0.014), whilst similar in participants since onset of symptoms (87 participants, p=0.15) (S8). Many articles reported intermittent false negative RT-PCR test results for participants within the monitoring time span. Where participant viral loads were reported, several different profiles were distinguished; two examples are shown in Figure 6 13, 14 . Intermittent false negative results were reported either where the level of virus is close to the limit of detection, or in participants with high viral load but for unclear reasons. The proportion of studies with high, low, or unclear ROB for each domain is shown in Figure 7 and ROB for individual studies is shown in S5. All studies were judged at high ROB. All but one were judged at high ROB for the participant selection domain 16 , mainly as they only included participants with confirmed SARS-CoV-2 infection based on at least one positive PCR test. Studies also frequently selected a subset of the participant cohort for longitudinal RT-PCR testing, and only results for these participants were included in the study. Ten studies were judged at unclear ROB for the index test domain as the schedule of testing was based on clinician choice rather than being pre-specified by the study or clinical guidelines, or because the samples used for PCR testing were not pre-specified. Eleven studies were judged at high ROB for the flow and timing domain mainly because continued testing was influenced by easy access to participants, such as by continued hospitalisation. Negative RT-PCR test results were common in people with SARS-CoV-2 infection confirming that RT-PCR testing misses identification of people with disease. Our IPD systematic review . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . has established that sampling site and time of testing are key determinants of whether SARS-CoV-2 infected individuals are identified by RT-PCR. We found that nasopharyngeal sampling was positive in approximately 89% (95% CI 83 to 93) of tests within 4 days of either symptom onset. Sampling ten days after symptom onset greatly reduced the chance of a positive test result. There were limited data on new methods of sample collection like saliva in these longitudinal studies. Sputum samples have similar or higher levels of detection to nasopharyngeal sampling, although this may be influenced by preferential sputum sampling in severely ill participants. Although based on few participants tested at both sampling sites, URT sites have faster viral clearance than LRT in most of these participants; 50% of participants were undetectable at URT sites 12 days after symptom onset compared to 28 days for LRT. We found that faecal sampling is not suitable for initial detection of disease, as up to 30% of participants detected using respiratory sampling are not detected using faecal sampling. All included studies were judged at high ROB so results of this review should be interpreted with caution. Box 1 provides an overview of the major methodological limitations and their potential impact on study results. A major source of bias is that all but one study 18 restricted inclusion to participants with confirmed SARS-CoV-2 infection based on at least one positive RT-PCR test, meaning that the percentage of positive RT-PCR testing is likely to be overestimated. Lack of technical details, for example of how samples are taken and RT-PCR tests performed, limit the applicability of findings to current testing. Compared to real life, studies were likely to use more invasive sampling methods, use experienced staff to obtain samples, and sample participants in hospital settings where sample handling could be standardised. Consequently, estimates of test performance are likely to be overestimated compared to real-world clinical use and in community population testing including self-test kits. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . This review uses robust systematic review methods to synthesise published literature and identifies overall patterns not possible from individual articles. Using IPD, we examined data across studies and avoided study level ecological biases present when using overall study estimates. IPD regarding sample site at different timepoints during infection is vital because it provides an overview of test performance impossible from individual studies alone. Synthesised IPD can also substantiate or reject patterns appearing within individual studies. Within participant paired comparisons of sampling sites also becomes possible with sufficient data. The main limitation is the risk of bias in the included studies. Although constraints were understandable given the circumstances in which the studies were done, the consequences for validity need to be highlighted. The percentage of positive RT-PCR testing is likely to be . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . overestimated, because inclusion was restricted to participants with confirmed SARS-CoV-2 infection based on at least one positive RT-PCR test in all but one study 18 Poor reporting of sampling methods and sites impaired our ability to distinguish between and report on variability between them. For some sampling methods such as saliva and throat swabs, more studies are needed. There were also sparse data on sampling methods that are becoming more widespread, such as participant self-sampling 41 and short nasal swab sampling (anterior nares/mid turbinate) 42 . Our index times may be subject to bias as symptom onset is somewhat subjective and hospital admission practices vary by country, pandemic stage, and hospital role (i.e. healthcare vs. isolation). The results presented do not correspond to following the same participants across time, but the testing at clinically relevant time snapshots reported from individual studies, so that participants tested at later time points are likely to have more severe disease; this does not limit the interpretation of results in understanding testing of participants in most clinical contexts. Comparisons of sampling sites should be restricted to participants tested at the relevant sites. We have used analysis methods that do not include clustering within studies, to keep analyses simple to understand and present, and to avoid complications of fitting models where the number of participants in each cluster varies. Ultimately, many potentially eligible studies did not report IPD which led to their exclusion, or only reported IPD for a subset of participants in the study. We would welcome contact and data sharing with clinicians and authors to rectify this. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . To avoid the consequences of missed infection, samples for RT-PCR testing need to be taken as soon as symptoms start for detection of SARS-CoV-2 infection in preventing ongoing transmission. Even within four days of symptom onset some participants infected with SARS-CoV-2 will receive negative test results. Testing at later times will result in a higher percentage of false negative tests in people with SARS-CoV-2, particularly at upper RT sampling sites. After ten Further sharing of IPD will be important and we would welcome contact from groups with IPD data we can include in ongoing research. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07.13.20152793 doi: medRxiv preprint Figure 6A show a participant where virus levels have reduced over time to a level around the limit of viral detection, and at these low levels of virus intermittent negative results will occur due to differences in the location or amount of sample. Figure 6B shows an example of a participant with high viral load, but where alternate RT-PCR test results report high viral load or undetectable virus. An adapted version of QUADAS-2 for longitudinal studies was used (Supp_table 3). For each domain, the percentage of studies by concern for potential risk of bias is shown: low (green), unclear (yellow) and high (red). . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . https://doi.org/10.1101/2020.07.13.20152793 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . Details of bias and applicability issues Impact on interpretation of study data Participants (source of bias) In these studies, the reference test usually incorporates RT-PCR (index test)  RT-PCR testing is usually a key component of identifying people with SARS-CoV-2 infection  Participants will not be detected or included in these studies when SARS-CoV-2 is not present at easily sampled sites and at the time that participants were available for testing Rates of positivity will be inflated as only people with virus accessible for sampling for RT-PCR tests will be included in studies Participants (source of bias) Most participants are identified or present based on respiratory tract symptoms such as cough or respiratory distress. Unclear how many and what severity of participants with SARS-CoV-2 are not included in studies  Participants will not be detected or included in these studies when less common symptoms or asymptomatic  Participants included will be biased to over-represent people with detectable virus in respiratory tract sampling sites and at times frequently used for testing (post symptom onset or at admission to hospital) Studies will inflate positivity for sampling sites that overlap with sampling sites used in RT-PCR reference testing  For example, we identified 30% of participants with RT positivity but with negative results from faecal sampling. However, if participants had only faecal virus, would they have been included in the studies? Index test: RT-PCR (applicability)  Studies included are likely to use more invasive sampling methods than acceptable in widespread population testing. For example, nasopharyngeal testing is likely in many current studies to be based on long swabs and self test kits Percentage of people with detectable virus may be overestimated when testing is applied in real world clinical use and in population testing  Studies will use experienced staff to obtain samples, handle, process and conduct tests  Studies are mostly sampling participants in hospital settings or in specialised research community testing research where sample handling, transport and storage have been standardised . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. Reporting of sampling sites and methods is poor  Poor reporting may have led to less ideal grouping of sampling in analysis  Some studies are likely to use a variety of nasopharyngeal sampling methods depending on the individual participants, but the type of sampling is typically reported at a study level for a particular sampling site Percentage of people with detectable virus may be over or underestimated Uncertainty and inconsistencies in time of sampling Percentage of people with detectable virus may be over or underestimated at particular times  Time of symptom onset can be subjective unless based on fever, but some participants do not have fever.  Time of symptom onset may be different if asked of participants in ICU setting  Time of hospitalisation and discharge may be affected by function hospitalisation serves in containment of disease spread. In some studies, the hospitals were also quarantine centres, so participants were hospitalised immediately at onset of mild symptoms rather than restricted to patients needing oxygen. Clinical cohort within studies changes across time points Percentage of people with detectable virus may be overestimated at particular later time points as these correspond to participants who were severely ill . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 14, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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