key: cord-0833378-2agqonyp
authors: Howard-Jones, Annaleise R.; Maddocks, Susan; Basile, Kerri; Dwyer, Dominic E.; Branley, James; Kok, Jen
title: Prolonged PCR positivity in elderly patients infected with SARS-CoV-2
date: 2021-09-23
journal: Pathology
DOI: 10.1016/j.pathol.2021.08.004
sha: b06eb452bece6bfecc76b0f2bc1f807251b14a3c
doc_id: 833378
cord_uid: 2agqonyp

nan

The global coronavirus disease 2019 (COVID-19) pandemic has raised many questions around the transmission dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Among these, defining the period of infectivity and, critically, proving clearance of the virus from the respiratory tract are paramount to public health efforts.

Reverse transcriptase polymerase chain reaction (RT-PCR) assays are now commonplace in diagnostic laboratories, and their limitations are well recognised. Clinicians are aware that common acute respiratory viral infections may be followed by prolonged upper respiratory tract PCR positivity. PCR assays specifically detect viral genetic material but typically cannot distinguish between intact replication-competent virus and residual, non-viable viral fragments. Recent studies have demonstrated that SARS-CoV-2 RNA may be detected well beyond the period in which viable virus can be cultured. [1] [2] [3] Shedding of culturable SARS-CoV-2 has been seen up to a median of 9 days from illness onset, 3 with PCR positivity persisting for a median of 17 days, longer in the upper respiratory tract than in the lower airway. 2 In our own centre, viable virus is more likely to be isolated in patients with more severe disease and with lower PCR cycle threshold (Ct) values, with no virus cultured from respiratory samples with a Ct >32. 1 more than 14 days from symptom onset were included. Date of symptom onset and dates of each positive sample were recorded. Duration of PCR positivity was defined as number of days from symptom onset until final positive SARS-CoV-2 PCR result.

All samples for SARS-CoV-2 testing were analysed as previously described. 1 In brief, samples were processed within 12 h of receipt in the laboratory. RNA was extracted from 200 µL of sample into an elution volume of 100 µL using the MagNA Pure 96 instrument's Viral NA Small Volume Kit (Roche Diagnostics, Germany) as per the manufacturer's instructions. SARS-CoV-2 nucleic acid amplification and detection by RT-PCR were performed using the LightCycler 480 II System (Roche Diagnostics, Switzerland). SARS-CoV-2 gene targets included between one and four of the following genes: E, N, RdRp, M, ORF1ab and ORF1b. Samples were considered positive with a cycle threshold (Ct) value of ≤40 on any target; Ct values of positive samples were recorded.

Statistical analysis was performed using the R statistical package (https://www.rproject.org/), utilising Welch's two-sided t test, with a significance threshold of p<0.05.

A non-research determination for this project was granted by the Health Protection NSW as it was a designated communicable disease control activity.

Over the study period, 90,031 samples were received. Of these, 1372 samples tested positive for SARS-CoV-2 (1.5%), with 387 samples (0.43%) from 72 patients displaying prolonged PCR positivity beyond 14 days from the date of symptom onset.

There was a statistically significant trend towards higher Ct values of all tested targets over time with a mean Ct of 30.1±6.6 at >14 days from symptom onset and 27.2±5.5 at 4-14 days, compared to 23.8±3.8 in the first 3 days (both p<0.001) (Fig. 1) .

The median age of all participants was 51 years (range 14-102 years) ( Fig. 2A) . The proportion of participants aged 80 years or over with persistently positive PCR results increased over time, from 11% at week 3 to 50% at week 6. Similarly, the mean age of participants with persistent PCR positivity increased over time (50 years in week 3, 74.5 years in week 6) (Fig. 2B ). Compared to participants aged under 40 years, the mean duration of PCR positivity in patients aged ≥80 years was significantly prolonged (p=0.007) ( Table 1) ; no participants <40 years of age remained PCR positive beyond 33 days from symptom onset.

The number of participants with prolonged SARS-CoV-2 PCR positivity declined with time from symptom onset. Of those positive at 14 days after symptom onset, 51% were still positive at 3 weeks, 25% at 4 weeks, 14% at 5 weeks, 5.6% at 6 weeks and 1.4% at 7-9 weeks (Fig. 2C) . SARS-CoV-2 RNA was detected in one patient up to 62 days after symptom onset.

PCR testing for SARS-CoV-2 RNA has a key role in initial diagnosis of COVID-19 disease due to its high analytical sensitivity and specificity, however its role in the latter phases of the infection is unclear. In this case series of adults aged 14-102 years, prolonged PCR positivity for >14 days from symptom onset was seen in <1% of all samples, with persistence for up to 62 days from symptom onset. This is consistent with earlier studies demonstrating prolonged RNA shedding following acute COVID-19 infection. 4, 5 In keeping with the current results, one US case series reported persistent PCR positivity rates of 98%, 86%, 56%, 27%, 9% and 5% at 1-week intervals from 1-6 weeks following symptom onset, 6 suggesting consistency across geographies despite disparate COVID-19 epidemic curves.

Our data demonstrate that older age (≥80 years) is correlated with prolonged duration of PCR positivity compared to younger persons (<40 years), aligning with earlier meta-analysis data showing a positive correlation of pooled mean duration of PCR positivity with age. 2 This is further corroborated by recent studies showing age, 7-9 residence in long-term care facilities, 10 medical comorbidities (including hypertension, chronic kidney disease, hyperlipidaemia, obesity and coronary artery disease), 7 immunomodulatory therapy 8 and duration of symptom onset 9 are associated with prolonged PCR positivity. By contrast, studies have shown no correlation between duration of PCR positivity and disease severity or clinical outcomes. 9 In patients with prolonged PCR positivity of at least 2 weeks from symptom onset, a significant trend in Ct toward higher values was seen with increasing time from symptom onset (Fig. 1) . These results, from a large cohort comprising hospitalised and nonhospitalised patients, are consistent with data from symptomatic non-hospitalised patients which also showed a trend toward higher Ct values over time. 11 As PCR assays cannot distinguish between nucleic acid detected from replicating virus and incomplete viral genetic fragments, prolonged PCR positivity may be expected beyond the acute infectious period. However, the pathophysiology underpinning the association with older age remains cryptic. Several possibilities exist, including a contribution from immunosenescence, 12 leading to impaired immune clearance of viral RNA in older people. Whether this association of age with prolonged PCR positivity is generalisable to novel variants of SARS-CoV-2 (including B.1.317.2) or indeed to other common respiratory viruses also warrants further investigation. Nucleic acid testing should not be used as a proof of cure in COVID-19. Small cohort studies have demonstrated no transmission events to household contacts of patients with prolonged PCR positivity, at least beyond 4 weeks from symptom onset, 13 and clearance of culturable virus occurs many days to weeks prior to cessation of PCR positivity. 3 Awaiting SARS-CoV-2 PCR negativity may unnecessarily delay de-isolation, particularly in older patients, where symptom resolution (and/or viral culture when available) provide more definitive evidence of the end of the infectious period. Larger studies are welcomed linking communitybased contact tracing with prolonged PCR positivity to further inform public health policies. 

Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19

SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis

Determining the communicable period of SARS-CoV-2: a rapid review of the literature

Temporal dynamics in viral shedding and transmissibility of COVID-19

Duration of viral shedding of discharged patients with severe COVID-19

Clinical predictors and timing of cessation of viral RNA shedding in patients with COVID-19

Comorbidities and age are associated with persistent covid-19 PCR positivity

Determinants of prolonged viral RNA shedding in hospitalized patients with SARS-CoV-2 infection

The effect of prolonged pcr positivity on patient outcomes and determination of isolation period in COVID-19 patients

Duration of SARS-CoV-2 shedding: a populationbased, Canadian study

SARS-CoV-2 viral load dynamics and real-time RT-PCR cycle threshold interpretation in symptomatic non-hospitalised individuals in New Zealand: a multicentre cross sectional observational study

Targeting aging cells improves survival

Prolonged presence of viral nucleic acid in clinically recovered COVID-19 patients was not associated with effective infectiousness

We gratefully acknowledge the work of the laboratory scientists of Institute of Clinical Pathology and Medical Research -NSW Health Pathology, Westmead, Australia.

This research did not receive any specific funding from the public, commercial, or not-for-profit sectors. The authors state that there are no conflicts of interest to disclose.