Elsevier

Pathology

Volume 53, Issue 4, June 2021, Pages 530-535
Pathology

VIROLOGY
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

https://doi.org/10.1016/j.pathol.2021.01.007Get rights and content

Summary

We conducted a multicentre cross sectional observational study of laboratory, public health and hospitalisation data for PCR-confirmed COVID-19 cases within the New Zealand Northern Region, between 12 February and 8 June 2020. The aim of this study was to describe population level SARS-CoV-2 upper respiratory tract (URT) viral load dynamics by stratifying positivity rates and polymerase chain reaction (PCR) cycle threshold (Ct) values of URT samples from COVID-19 cases by days since symptom onset, and to explore utility of Ct values in determining length of time post-infection and thus potential infectivity.

Of 123,124 samples tested for SARS-CoV-2 by PCR, 579 samples (407 positive and 172 negative) from 368 symptomatic non-hospitalised individuals with PCR-confirmed infection were included. Sample positivity rate was 61.5% (8/13) for pre-symptomatic samples, rising to 93.2% (317/340) for samples collected during the purported symptomatic infectious period (days 0–10 post-symptom onset), and dropping to 36.3% (82/226) for post-infectious period samples (day 11 onwards). URT viral load peaked shortly after symptom onset, with median Ct values ranging 20.00–29.99 until 15 days post-symptom onset, and >30.00 after this time. Of samples with a Ct value of <20.00, 96.1% were collected during the symptomatic infectious period. However, of samples with a Ct value ≥30.00 and ≥35.00, 46.9% and 18.5%, respectively, were also collected during the symptomatic infectious period.

The findings of this study indicate that at or soon after symptom onset represents the optimum time to test for SARS-CoV-2 in the URT, with median Ct values suggesting the useful testing window extends until around 15 days post-symptom onset. In asymptomatic individuals or those with unknown dates of symptom onset, Ct values <20.00 imply recent onset/potential infectivity, but Ct values ≥30.00 or ≥35.00 do not exclude recent onset/potential infectivity. Individual sample Ct values should not be used as an absolute marker of length of time post-infection or to exclude infectivity where date of symptom onset is unavailable.

Introduction

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reached New Zealand on 28 February 2020 when the country identified its first case.1 Following a rapid increase in cases the New Zealand Government declared a state of national emergency and mandated a nationwide social lockdown on 25 March (‘Alert Level 4’).1 This lockdown formed part of an outbreak elimination strategy designed to prevent importation of new cases and halt viral transmission within the populace. An essential element of such a strategy is rapid detection of new cases through widespread testing.1 The mainstay of COVID-19 testing consists of analysing clinical samples for the presence of SARS-CoV-2 nucleic acid using real time reverse transcription polymerase chain reaction technology (hereafter referred to as PCR).2 When a PCR test for SARS-CoV-2 detects the specified nucleic acid target sequences, a positive result is associated with a cycle threshold (Ct) value: the number of PCR cycles needed to produce a detectable signal.3 The lower the Ct value, the fewer PCR cycles needed to produce a positive result, the greater the quantity of viral nucleic acid in the sample tested, and the greater the quantity of viral nucleic acid (the viral load) in the anatomical site sampled.3 Ct values do not equate to direct viral load measurement (which requires standardisation using a reference curve) but provide a useful surrogate measure of viral load.4

In the initial stages of SARS-CoV-2 infection there is a high viral load in the upper respiratory tract (URT), with URT samples favoured for detecting early infection.2 Viral load in the URT decreases with time, however studies examining URT temporal viral load dynamics have primarily involved hospitalised patients,5 and viral dynamics in non-hospitalised individuals require further elaboration.

The infectious period is generally regarded as ending at 10 days post-symptom onset, with replication competent virus rarely isolated after this time.6, 7, 8 Increased screening of asymptomatic individuals (such as case contacts or returning travellers) poses a diagnostic and public health challenge, as with no clear date of symptom onset the infectious period cannot be easily delineated. This leads to the question of whether Ct values from positive results can predict infectivity and inform duration of isolation/quarantine.9 Specifically, does a higher Ct value (e.g., ≥35.00) imply that the infectious period has passed?

In this multicentre cross sectional observational study, we reviewed laboratory, public health and hospital admission data for all PCR-confirmed SARS-CoV-2 cases of all ages within the 1.75 million population of the Auckland and Northland areas of New Zealand [the Northern District Health Board (DHB) Region].10 This study had two main aims. Firstly, to describe SARS-CoV-2 URT viral load dynamics at a population level by comparing positivity rates and Ct values of samples from PCR-confirmed cases with time since symptom onset. A greater understanding of URT temporal viral load dynamics in symptomatic non-hospitalised individuals will enhance understanding of the testing window and interpretation of results in this group. Secondly, to describe the distribution of sample collection (split by purported symptomatic infectious period, days 0–10) in samples with given Ct value ranges. This will allow better understanding of the utility of Ct values in determining length of time post-infection and thus potential infectivity in the absence of a date of symptom onset.

Section snippets

Laboratory data

Four laboratories are routinely responsible for SARS-CoV-2 PCR testing within the Northern DHB Region of New Zealand: LabPLUS Auckland City Hospital, Auckland DHB (Site A); Labtests, Community Pathology Services Provider (Site B); Middlemore Hospital, Counties Manukau DHB (Site C); and North Shore Hospital, Waitematā DHB (Site D). A retrospective review of laboratory data was undertaken for all SARS-CoV-2 PCR tests performed by these sites between 12 February 2020 (the date SARS-CoV-2 PCR

Results

Between 12 February and 8 June 2020 inclusive, a total of 123,124 samples underwent SARS-CoV-2 PCR testing by the four study sites. Of the total number of samples tested, 122,628 (99.6%) tested negative (or indeterminate) and 496 (0.4%) tested positive. Taking positive and negative samples from individuals with PCR-confirmed SARS-CoV-2 infection, 708 samples were assessed for study inclusion. Subsequently, 129 samples were excluded, leaving 579 samples [407 (70.3%) positive and 172 (29.7%)

Discussion

In this study of symptomatic non-hospitalised individuals with PCR-confirmed COVID-19 in New Zealand, we examined population level results, including Ct value trends relative to time since symptom onset, to provide a surrogate measure of the URT viral load dynamics in mild SARS-CoV-2 infection.

We found that the positivity rate of samples differed by time since onset of symptoms, with approximately 60% of samples positive when collected in the pre-symptomatic period, rising to >90% of samples

Conclusion

In this study of SARS-CoV-2 temporal viral load dynamics in symptomatic non-hospitalised individuals with COVID-19 in New Zealand, we have shown that viral load peaks shortly after symptom onset, sample positivity rate is highest during the symptomatic infectious period (days 0–10), and median Ct values indicate that PCR is likely to be reliable for detecting SARS-CoV-2 infection in the first 15 days post-symptom onset, but diagnostic yield may drop after this time. Samples with Ct values

Conflict of interests and sources of funding

The authors state that there are no conflicts of interest to disclose.

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