Virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR

Virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR. Jefferson T, Heneghan C

https://www.cebm.net/study/virological-characterization-of-covid-19-patients-that-test-re-positive-for-sars-cov-2-by-rt-pcr/

Published on September 1, 2020

Reference Lu J, Peng J, Xiong Q, Liu Z, Lin H, Tan X, et al. Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR. EBioMedicine. 2020;59. 2020
Study type
Country Guangdong, China
Setting Hospital/community - Guangdong COVID-19 surveillance system
Funding Details Guangdong Provincial Novel Coronavirus Scientific and Technological Project, China Evergrande Group and Science and Technology Planning Project of Guangdong National Science and Technology Project
Transmission mode Viral load, Multiple
Exposures

Bottom Line

“Re-positive” cases are unlikely to be infectious as no intact RNA single helix was detected or live virus isolated. “Re-positive” discharged cases are caused by intermittent shedding of cells containing remnant RNA.

Evidence Summary

87 cases (14%) re-tested as positive for SARS-CoV-2 RNA via RT-PCR and returned to the local designated hospital for isolation and medical observation. 

Detection of virus RNA was observed in all age groups (3 months to 69 years, median age of 28 years, much younger than in Guangdong (median age of 47 years). 

All “repositive” cases had only mild (46) or moderate (41) clinical symptoms during initial hospitalization. No “re-positive” cases had been classified as severe and were re tested a median of 14 days post discharge, which was shorter than the severe cases ( median 7 days). 

58 of 59 tested (98%) “re-positive” cases developed Neutralising Antibodies with a titre >4, ranging from 4 to >1024 showing competent immune activation in re-positive cases. 

137 swabs (51 nasopharyngeal, 18 throat and 68 anal), were tested with three different PCR kits to assess possible differences between kits (hypothesis 4 below) and reduce false negatives. 

36 swabs (14 nasopharyngeal, 3 throat and 14 anal swabs) from 33 cases were positive by at least one RT-PCR kit and inoculated into Vero E6 cells.

Out of 619 discharged cases, 14% were SARS-CoV-2 “re-positive” cases 

As all dischargees were strictly quarantined and isolated the “repositives” are unlikely to be due to re-infection.

All “repostive” cases were younger and had milder symptoms and shorter onset to discharge times than the average COVID-19 cases (onset-discharge 17 (7-36) vs 33 days (8-66)). 

It is also unlikely that cases were discharged too early because they were only originally discharged after two negative nasopharyngeal and anal swabs.

The observation of “re-positive” SARS-CoV-2 RNA cases is not random and mainly observed in young cases without severe clinical symptoms.The observation of “re-positive” SARS-CoV-2 RNA cases is not random and observed in young cases without severe clinical symptoms.

None of the cultures were positive but 58/59 respostives had neutralising antibodies showing immune reaction to the virus and explaining that “repositives” have little infectious potential. 

Of note that no complete RNA genome sequence was isolated, only up to  a maximum of 75.48% of genome suggesting high degradation of viral particles.

Discrepancy between PCR test kits

Even more important there was a discrepancy between kits: some produced sequences that covered < 10% of the virus genome while 12 samples detected as negative by three RT-PCR kits gave rise to virus sequences that spanned >10% of the virus genome. 

This points to diversity in hit rates between different kits with different primers targeting different parts of the genome, in the presence of an incomplete genome.

Genome coverage was higher in fecal than respiratory samples.

Figure 3a in the paper shows sequencing by kit, probed gene and ct. Cts vary from 29 to 39 across the board but also by kit for the same specimen and the same targeted gene.

What did they do?

The study assessed the SARS-CoV-2 “repositive” cases out of 619 discharged COVID-19 cases between 23 January and 19 February, in Guangdong, China. 

The ultimate aim was to assess the link between infectivity and molecular testing.

The authors investigated the reasons for testing positive again for SARS CoV-2 after hospital discharge. They report 4 possible reasons for such a finding:

  1. relapse or recrudescent infection with the “first” SARS-CoV-2 
  2. re-infection with a “second” SARS-CoV-2 inoculum
  3. remnant RNA fragments of the “first” SARS-CoV-2 infection, resulting from intermittent shedding of cells containing viral fragments
  4. laboratory errors, or technical limits of RT-PCR assay

To test these explanations the authors tested 137 swabs and 59 serum samples from 70 “repositive” cases to assess the immunological and virologic characteristics of the SARS-CoV-2 “repositive” cases. From 23 January, hospital dischargees followed a strict isolation protocol living (for example) in single dedicated hotel rooms and went home only when nucleic acid tests were negative on both respiratory tract and digestive tract samples. Samples (nasopharyngeal, throat and anal swabs), were collected for RT-PCR diagnosis at 7 days and 14 days after discharge. 

Culture was carried out by inoculating vero E6 cells with patient sample. Cytopathic effect (CPE) were observed daily but if no CPE was observed at 7 days, supernatant was collected and used for the second round of passage. 

RT-PCR diagnosis was carried out on RNA using three RT-PCR kits to conduct nucleic acid testing, in an attempt to avoid false negatives.

Study reliability

The study is a well thought out and well reported seminal study. The only problem is the lack of consecutive re-testing of samples from the same patients which may have affected some of the time periods reported in Table 1 of the study (see case flow diagram).

Clearly defined setting Demographic characteristics described Follow-up length was sufficient Transmission outcomes assessed Main biases are taken into consideration
Yes Yes Yes Yes Partly

What else should I consider?

This results of this study require confirmation.

About the authors

Carl Heneghan

Carl Heneghan

Carl is Professor of EBM & Director of CEBM at the University of Oxford. He is also a GP and tweets @carlheneghan. He has an active interest in discovering the truth behind health research findings

Tom Jefferson

Tom Jefferson

Tom Jefferson is a senior associate tutor and honorary research fellow, Centre for Evidence-Based Medicine, University of Oxford.