Re-positive of SARS-CoV-2 test is common in COVID-19 patients after hospital discharge. Data from high standard post-discharge quarantined patients in Macao SAR, China

Introduction

In December 2019, a series of pneumonia cases of novel coronavirus infection emerged in Wuhan city of Hubei province in China. Then, the disease spread quickly and widely worldwide and was named COVID-19 (Li et al., 2020). As a rising number of the issue, the WHO declared the outbreak as a Public Health Emergency of International Concern on 30 Jan 2020 (World Health Organization, 2020a). Up to early November, the accumulative number of confirmed cases has reached over 46 million worldwide, and it continues to rise (World Health Organization, 2020b). Some studies even found the Spike D614G mutation of the virus with increased infectivity in other countries while comparing the virus samples in China (Korber et al., 2020). As a famous gambling and tourism city with the highest population density globally, Macau faces massive stress from overseas and local populations during the coronavirus pandemic. From our point of view, Macau fights against COVID-19 quite successfully. There were only 45 confirmed cases in Macau’s two irrelevant outbreaks before late June, without local transmission. All ten patients in the first outbreak from 22 Jan to 4 Feb were from Mainland China, while the 35 cases in the second episode from 15 Mar to 8 Apr came from foreign countries. After that, there were no more new cases for over two months (Macao SAR Government Portal, 2020). The Macau Government’s tremendous effort contributed to the great success in this fighting.

We made early upgrades to our port policy to a 14-d quarantine with at least twice RT-PCR tests of novel coronavirus for all people who came from other countries since March (Lio et al., 2020). On the other hand, we had a loose community testing policy, making RT-PCR tests accessible to everyone whenever they requested. Under these aggressive public health strategies, we could safely confirm all infected cases in the second outbreak.

Meanwhile, studies reported SARS-CoV-2 re-positive cases after the patients fulfilled discharge criteria (Lan et al., 2020; Yuan et al., 2020; Zou et al., 2020; Wang et al., 2020). It raised concerns about the virus’s re-infection or reactivation, regardless of whether there was no substantial evidence about their infectivity. Viewing this, we quarantined all patients who fulfilled discharge criteria in airborne infection isolation rooms, for at least 14 days, with RT-PCR test on day 10 and day 13, before they were back to the Macau community. The re-positive rate in academic reports was 3.3% to 21.4% (Yuan et al., 2020; Zou et al., 2020; Wang et al., 2020; Kang, 2020; Xiao, Tong & Zhang, 2020; Justin et al., 2020). Most of their patients became infected in China. Some were home isolated while some used inconsistent novel coronavirus test methods and other surveys represented only partial cases in a city. Here, we reported the re-positive rate of all COVID-19 patients in Macau during the second outbreak. All of them were infected outside of China and observed under a high standard quarantine location with repeated nasopharyngeal swab RT-PCR tests after hospital discharge.

Materials & Methods

Results

Discussion

One-third of patients had re-positive SARS-CoV-2 test results in our study, which is higher than other studies (Yuan et al., 2020; Zou et al., 2020; Wang et al., 2020; Kang, 2020; Xiao, Tong & Zhang, 2020; Justin et al., 2020). Under the outstanding public health strategies and an ideal environment in Macau, we could evaluate the re-positive rate of all the patients quarantined in a high standard location with RT-PCR tests after hospital discharge within one independent outbreak. After all the serious intervention and observation, we could conclude that a significant proportion of discharged patients in Macau were carriers of the virus nucleic acid. Because they were all imported cases in our study, we suspected this phenomenon might be shared globally.

The re-positive rate in our study was as high as 34.3%. A more significant number of tests may be one possible reason. We performed twice tests while the other reported countries only had once (Zou et al., 2020; Wang et al., 2020; Kang, 2020; Xiao, Tong & Zhang, 2020; Justin et al., 2020). The probability of obtaining a false-negative result increased with time from symptom onset and could be drastically reduced by repeat testing (Wikramaratna et al., 2020). In previous studies, the heterogenic design, case selection, and various sampling sites and frequency led to a relatively doubtful re-positive rate (Yuan et al., 2020; Zou et al., 2020; Wang et al., 2020; Kang, 2020; Xiao, Tong & Zhang, 2020; Justin et al., 2020). In contrast, our result should provide more accurate data since we assessed all cases in an outbreak with a unified approach.

Apart from false-negative results, false-positive tests should be considered another likely reason for the inaccurate re-positive rate. The primary route of false-positive PCR test is contamination, including cross-contamination between specimens or synthetically derived target nucleic acids (Huggett et al., 2020). All our specimens were performed in an ISO 15189 accredited medical laboratory. Therefore, the likelihood of contamination should be low, and subsequently the false-positive cases.

Along with the global dispersal of COVID-19, mutant viruses have emerged and lead to alteration of the virus behavior. The Spike protein amino acid change D614G is one of the most concerned SARS-CoV-2 variants, which has become apparent in Europe and rapidly replacing other versions of the virus globally since late February, except in China (Korber et al., 2020).Different from most existing research, all cases in this study were from foreign countries and might be infected by the D614G variant. One recent research found that the D614G replacement was associated with higher viral loads (Volz et al., 2021). It can be an explanation of the unexpected high re-positive rate.

Our study showed a trend that symptomatic patients were more likely to be re-positive. It also found that the median days from S/S onset to diagnosis was shorter in the re-positive group than the negative group, but the duration from diagnosis to hospital discharge was the opposite. However, their statistical significance could not be evaluated due to the small sample size. This result reflected that re-positive patients were more likely to have an earlier diagnosis and more extended hospitalization than the negative group. In other words, the symptomatic and early diagnosed patients tended to be re-positive during the quarantine period. Can there be a higher virus load in vivo cells in this group of patients, with more pronounced symptoms and signs that make a more straightforward diagnosis? Further study is worth to clear this issue.

Although several studies analyzed the risk factors of recurrent positive SARS-CoV-2 test among discharged patients (Yuan et al., 2020; Zou et al., 2020; Wang et al., 2020; Xiao, Tong & Zhang, 2020; Justin et al., 2020), there was no unanimous conclusion till the present moment. Some research found the re-positive result related to different risk factors such as age, symptoms, illness severity, laboratory tests, etc. Still, they had the opposite contribution in other reports. Indeed, none of the present studies could discover reliable indicators to forecast the patient’s risk of being re-positive SARS-CoV-2. Our analysis did not establish any risk factors in terms of demographic or clinical characteristics, laboratory examination, or treatment strategies. As a high re-positive rate was noted and failure to discover risk factors, should re-positive be a natural pathophysiological process of COVID-19?

Except for laboratory error, the causes of a re-positive SARS-CoV-2 test result after hospital discharge have several assumptions. They are re-infection, reactivation, and residual virus nucleic acid.

Hong Kong experts confirmed the possibility of re-infection in COVID-19 patients in a recent study (To et al., 2020). It could be one reason for the re-positive test in the previous academic reports. Unclarified or home isolation strategy could imply a little or some opportunity for the people to be exposed to the infection source again. Our research quarantined all patients in airborne infection isolation rooms and under management by professional medical staff with suitable personal protective equipment. Meanwhile, there was no infection report in medical staff and no local infection case in Macau. As a result, we can exclude the chance of re-infection in this high standard environment.

Reactivation is unlikely since all our re-positive cases were asymptomatic during post-discharge quarantine, and they already had IgG antiviral antibodies in serum samples before discharge. After deducting the above hypotheses, residual virus nucleic acid in vivo cell is a possible cause of the re-positive phenomenon.

Liu WD and colleagues (2020) reported a COVID-19 case with prolonged virus shedding even after seroconversion. It implied that the SARS-CoV-2 could exist in human cells in vivo for an extended period. On the other hand, Justin W and colleagues observed the oscillation of positive and negative SARS-CoV-2 NPS test results in the patients, which related to a fluctuating cycle threshold (Ct) value. Therefore, we presume virus load would descend along with the disease’s recovery, with a relative up and down pattern rather than in a straight line. When infected cells were intermittently shedding at the level above and below the detection limit, conversion between positive and negative results would appear. Once we conducted the test at the point of fewer shedding cells, the result would be negative, and the patient would fulfill the discharge criteria. Actually, the virus shedding load just hit the detection limit, and more cells would be passed at the other time, then the patient would have a re-positive test result. Of course, further precise and quantitative experimental studies are needed to verify this hypothesis.

It is crucial to clarify the infectivity in re-positive patients clinically and in public health decision making. However, it remains unknown despite substantive reports and research. The virus’s ability to replicate in cultured cells serves as a surrogate marker of infectivity, but we did not perform virus culture due to technology limitations. On the other hand, culture is less sensitive than RT-PCR for detection of live viruses. Even with the culture of virus, mixed results were reported in different studies. Wölfel R and colleagues (2020)found that no virus could be cultured in patient samples after day 9 of symptom onset. In contrast, Liu WD and colleagues reported isolated SARS-CoV-2 from the sputum sample on day 18 after symptoms onset (Liu et al., 2020). Therefore, there is still no applicable clinical method to establish SARS-CoV-2’s infectivity. The potential risk of virus transmission from a re-positive patient cannot be eliminated.

In Macau, most of our citizens are susceptible to COVID-19 since there is no local epidemic, and only very few residents were infected. Fortunately, enough resources such as AIIR, PPE, healthcare human resources, etc., which allow high quality and prolonged quarantine and minimize transmission risk in our city. After balancing the risk and benefit, we released re-positive patients from quarantine after three consecutive negative results. Zou Y and colleagues also supported that patients with three consecutive negative results had a much lower recurrent positive rate than two negative findings (Zou et al., 2020).This public health strategy contributes to Macau’s achievements of beating back COVID-19. However, it did bring some undesired experience to the individuals. For example, prolonged isolation in a health facility is prone to psychological problems. As a result, other cities may not simply copy our methods. The health authority should make their decision according to community acceptance ability. Nonetheless, we agreed that quarantine at home for two weeks with PCR tests after hospital discharge is the least transmission prevention since SARS-CoV-2 is a novel virus with only scanty knowledge about it and no effective treatment until now.

Lastly, some limitations of this study should be noted. First, the sample size was relatively small. There were only 35 cases in our study, even though all patients in Macau’s second outbreak had been included. Second, the onset time and the presentation of sign or symptom was according to the patient’s statement so that recall bias may present in the timeline of disease progression. Third, we did not perform the virus culture, and the infectivity could not be shown. Fourth, we could not get Ct values and genomic sequencing of SARS-CoV-2 due to laboratory policy. They should be considered in future studies.

According to the global COVID-19 situation, the disease seems to keep coexisting with humans for a long time. Since a high re-positive rate among COVID-19 patients was presented in our study, we can imagine that there will be a great practical challenge to handle this group of patients. Different underlying causes had different approaches. How can we distinguish re-infection, reactivation, and residual virus nucleic acid from each other? To do or not to do the whole-genome sequencing? This is a question. For the patients with residual virus nucleic acid, overtreatment may do some harm to individuals.

Conclusions

A re-positive SARS-CoV-2 test after having fulfilled discharge criteria is a common phenomenon. The patients diagnosed in the early stage of illness and those with extended hospitalization were more likely to have recurrent positive results. Virus re-infection is impossible in our report, and reactivation is also unlikely, so that residual virus nucleic acid should be a reasonable explanation for this phenomenon. As there is insufficient evidence to exclude infectivity in re-positive patients, it is better to quarantine patients after discharge for a prolonged period with repeated RT-PCR tests. It may minimize the community’s potential risk, particularly in regions with relatively plenty of resources and low community infection rates, such as Macau.

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