COVID-19 negative patients who retested positive

Abstract

Background: Research studies comparing antibody response from coronavirus disease 2019 (COVID-19) cases that retested positive (RP) using reverse transcription polymerase chain reaction (RT-PCR) and those who did not retest positive (NRP) were used to investigate a possible relationship between antibody response and retesting status.

Methods: Seven data bases were searched. Research criteria included cohort and case-control studies, carried out worldwide and published before September 9, 2020, that compared the serum antibody levels of hospitalized COVID-19 cases that RP after discharge to those that did NRP.

Results: There is some evidence that immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody levels in RP cases were lower compared with NRP cases. The hypothesis of incomplete clearance aligns with these findings. The possibility of false negative reverse transcription polymerase chain reaction (RT-PCR test results during viral clearance is also plausible, as concentration of the viral ribonucleic acid (RNA) in nasopharyngeal and fecal swabs fluctuate below the limits of RT-PCR detection during virus clearance. The probability of reinfection was less likely to be the cause of retesting positive because of the low risk of exposure where cases observed a 14 day-quarantine after discharge.

Conclusion: More studies are needed to better explain the immune response of recovered COVID-19 cases retesting positive after discharge.

Introduction

Coronavirus disease 2019 (COVID-19) is a novel disease that results from infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)^{Footnote 1}. On December 31, 2019, a case of pneumonia of unknown cause was identified in Wuhan, China and reported to the World Health Organization (WHO) Country Office. The cause of the disease was later confirmed to be a novel coronavirus^{Footnote 2}. The SARS-CoV-2 outbreak was declared a pandemic by WHO on March 11, 2020^{Footnote 3}. As of October 14, 2020, 75% of the 38,508,475 internationally confirmed cases of COVID-19 have recovered^{Footnote 4}. However, there is evidence that some recovered COVID-19 cases discharged from hospital with negative COVID-19 tests later retested positive (RP)^{Footnote 5}.

Current situation

The situation of recovered and discharged COVID-19 cases retesting positive raise concerns of potential reinfection or incomplete clearance of the virus, as well as questions related to a patient’s infectiousness. Multiple hypotheses have been put forward to explain the reasons why some cases RP after discharge with negative reverse transcription polymerase chain reaction (RT-PCR) test results. The purpose of this review is to explore the current evidence regarding differences in antibody response between discharged COVID-19 cases that RP and those that did not retest positive (NRP).

Methods

Our research criteria included cohort and case-control studies carried out across the world and published before September 9, 2020, that compared the serum antibody level of hospitalized COVID-19 cases that RP after discharge to those that did not. Seven databases were searched. Search terms used included the following: reactivation; reinfection; reoccurrence; recurrent; in conjunction with hospitalization; discharge; antibody; and immunity. Articles (n=224) were screened for relevance and included studies were evaluated with the Newcastle-Ottawa Scale Risk of Bias Tool according to criteria related to the selection of the cases (score=4), the comparability of RP cases to NRP cases (score=2) and the assessment of the exposure or the outcome (score=3). A total of nine studies were found to be relevant (see Appendix A). A follow-up of at least four weeks was considered as adequate in cohort studies. The assessment was done by two reviewers (see Appendix B).

Results

We identified three prospective cohort studies^{Footnote 6Footnote 7Footnote 8}, three retrospective cohort studies^{Footnote 9Footnote 10Footnote 11}, two cohort studies (unclear if prospective or retrospective)^{Footnote 12Footnote 13} and one case-control study^{Footnote 14}.

The sample size of the nine studies varied from 74 to 619. The prevalence of COVID-19 cases that experienced a reoccurrence of a positive PCR test after meeting discharge criteria of two consecutive negative RT-PCR tests more than 24 hours apart, varied from 7% to 21% in seven out of eight cohort studies^{Footnote 6Footnote 7Footnote 8Footnote 10Footnote 11Footnote 12Footnote 13Footnote 14}, with the ninth study finding 58% RT^{Footnote 9}. The average prevalence for the eight cohort studies was 16.6% (n=382/2,305 cases). Of the six studies that captured information on the time between testing negative at discharge and RP the first time post discharge, the median time across studies varied from 4.6 days to 12 days ^{Footnote 6Footnote 8Footnote 10Footnote 11Footnote 13Footnote 14}. The RP cases were significantly younger in four studies^{Footnote 6Footnote 8Footnote 13Footnote 14}; however, no age difference was found in the five other studies^{Footnote 7Footnote 9Footnote 10Footnote 11Footnote 12}. No association with sex was reported in any study.

Six studies reported on the positivity rate of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies^{Footnote 7Footnote 8Footnote 10Footnote 11Footnote 12Footnote 13}, while four specified their serum levels^{Footnote 8Footnote 9Footnote 12Footnote 14}. There were no differences in IgG and IgM antibody positivity rates between RP and NRP cases^{Footnote 6Footnote 7Footnote 8Footnote 10Footnote 11Footnote 12Footnote 13}; however, the level of IgG^{Footnote 9Footnote 14}and/or IgM ^{Footnote 8Footnote 14} antibodies in the serum was lower in RP cases compared with NRP cases in three studies. No difference was observed in either IgM^{Footnote 9Footnote 12} or IgG level^{Footnote 8Footnote 12} in the other studies.

Discussion

There are several possible explanations for what a RP result following discharge such as low viral load in samples, false negative results in RT-PCR tests and re-infection. The most likely explanation is a false negative RT-PCR result due to 1) viral ribonucleic acid (RNA) concentration decreasing to levels below the limit of detection of the test during viral clearance and 2) the expected proportion of false negative results in RT-PCR tests.

According to Liu et al.^{Footnote 12}, given that the presence of IgM antibodies was similar in both cases who RP and NRP, the RP RT-PCR tests in their review of 619 cases were unlikely to be due to reinfection with SARS-CoV-2 virus. The studies included in this review mainly monitored cases during the post-discharge 14-day quarantine period, which would also rule out re-infection as a likely explanation for RP results shortly after discharge.

According to Yang et al., false negative RT-PCR results could also result from low virus concentrations in samples from nasopharyngeal or fecal swabs despite viruses still being present in the lower respiratory tract, leading to intermittent or fluctuating excretion of viral RNA in the upper respiratory tract^{Footnote 8}.

Wölfel et al. reported that the RT-PCR for SARS-CoV-2 is an imperfect test, with a sensitivity of 89% and, thus, an expected proportion of 11% false negative results^{Footnote 15}. Zou^{Footnote 11} found that performing three consecutive tests prior to discharge significantly reduced the chance of RP, which is consistent with the false-negative results hypothesis.
The findings that relate lower level of antibodies to RP suggest that cases that RP were more likely to have a weaker immune response, which aligns with the hypothesis of a viral clearance. According to Yuan^{Footnote 7}, the lowest prevalence of subsequent positive tests experienced by cases with severe symptoms compared with those experiencing mild symptoms can be explained by a stronger immune response activated in severely ill cases that clears the virus more effectively.

One of the main concerns that arise from cases RP is the infectiousness of cases after discharge. While some authors argued that the risk of infectiousness during this period is low^{Footnote 8Footnote 16}, others claimed the opposite and even mention the possibility of chronic infection with SARS-CoV-2^{Footnote 12}. This latter assumption casts doubt on the protective role of IgG antibodies and of using serology testing to establish immunity.

The findings of this review seemed to be supportive of an association between antibody response and RP after discharge. There is some evidence that IgG and IgM antibodies levels in RP cases are lower compared with NRP cases. The hypothesis of incomplete clearance also aligns with these findings.

Limitations

The current review has several limitations. Five out of nine studies had a sample size of less than 200 and all studies were restricted to China, which limits the representativeness of the review. We were unable to find research outside of China that had serology results, a comparison group and had follow-up RT-PCR testing to establish RP status. Among the included studies, the duration of follow-up after discharge and RT-PCR testing intervals varied, which could impact the results related to the timing and prevalence of RP results summarized in this study. In addition, eight out of nine studies were based on discharged cases but no information about their representativeness of hospitalized cases was provided. Further, the lack of comparison of cases with incomplete medical records or lost to follow-up to those that remained in the studies limits the assessment of potential bias estimates. This study included studies published up to September 2020 and should be interpreted accordingly, given the rapid evolution of the evidence. It could be valuable for future studies to focus on testing practices that could reduce the probability of false negative tests to ensure that hospitalized COVID-19 cases meet the required criteria before their discharge.

None of the studies examined the potential association in a multivariable analysis with antibody response to determine the adjusted associations after controlling for potential confounders.

Conclusion

The situation of COVID-19 cases subsequently RP for COVID-19 after having two negative RT-PCR test results is not uncommon. Evidence suggests a relationship between RP cases and age^{Footnote 6Footnote 8Footnote 13Footnote 14} and possibly between RP and disease severity. However, none of the studies examined the potential association in a multivariable analysis with antibody response to determine the adjusted associations after controlling for potential confounders. Additional evidence synthesis work with proper observational studies on the characteristics of COVID-19 cases that RP is needed to better understand who is likely to RP. Similarly, additional research and synthesis work on immune response and immunity is needed to improve our understanding of COVID-19 infection.

The evidence summarized in this report may have important implications for public health and management of recovered COVID-19 cases. There was a limited number of studies that met the inclusion criteria; however, the evidence suggests the immune response in convalescent COVID-19 cases may be associated with the incomplete viral clearance. These preliminary results can be used to inform further research or decision making on this topic.

Authors’ statement

• NA — Methodology, investigation, writing–original draft, review and editing
• MS — Methodology, investigation, writing–original draft, review and editing
• RE — Conceptualization, methodology, investigation, writing–original draft, review and editing, supervision
• CA — Writing–original draft, review and editing
• AL — Writing–original draft, review and editing
• LW — Writing–review and editing
• IGJ — Writing–review and editing
• ET — Writing–review and editing
• TD — Writing–review and editing

Competing interests

None to declare.

Acknowledgements

We would like to thank our colleagues at the Emerging Sciences Group at the Public Health Agency of Canada for their support on this work.

Funding

None to declare.

Appendices