The possibility of reinfection or reactivation of COVID-19 is concerning to the physicians and the patients. Articles that have reported these phenomena raise questions that when answered will have a pivotal impact on worldwide preventive and treatment policies. If reinfections occur, not only seemingly recovered patients can infect others, but also achieving herd immunity via natural infection or immunity after vaccination will be challenging (22).
Interpretations of a positive RT-PCR re-test
Considering the large number of people who have been diagnosed with COVID-19, few cases of reinfection have been reported. Often, for the diagnosis COVID-19 in a recovered individual a positive RT-PCR test with or without accompanying symptoms have been used. Although, in these cases different speculations for interpretation of a positive RT-PCR test can be made; each of which will have specific reverberations (23).
In some studies, a positive RT-PCR result in a recovering patient who tested negative upon resolution of their symptoms, is considered as a strong indicator of reinfection (10). Although, we need to take into account the possibility of a false result in either test, which would undermine a diagnosis of re-infection (24). A false positive test can occur in a recovering patient; or a false negative test shortly after subsidence of symptoms, followed later by a correctly positive test may mislead physicians towards a diagnosis of re-infection (25). To avoid this problem, as per WHO recommendations, in many regions the treatment protocol for COVID-19 requires two consecutive negative RT-PCR results prior to discharge (26, 27).
Also, RT-PCR as the main diagnostic tool of infection cannot distinguish between replication-competent viruses and remnants of viral fragments that are expelled from a recovering, non-contagious patient (28, 29). Viral shedding from the respiratory tract during the recovery period has been reported to last for as long as 12 weeks after the initial infection (30); thus, a significant time-gap between the first episode of infection and the second positive RT-PCR test can clarify that the patient has passed the post-COVID-19 viral shedding stage.
The first follow-up RT-PCR
In our study, as per national protocol, no RT-PCR testing was performed upon discharge to confirm viral clearance, and the first follow-up RT-PCR study was performed 30 days after the first positive RT-PCR result in each patient, which would have been after at least 14 days of being asymptomatic. Considering the short interval between the onset of the disease and the test, no recorded negative RT-PCR result at discharge, and the fact that none of the patients who tested positive were symptomatic, we believe that the positive results show continued viral shedding rather than re-infection. The four patients who tested positive were not significantly different from those who tested negative in terms of the severity of the initial illness, duration of the first admission, age, or significant underlying conditions to suggest a risk factor for a longer viral shedding period. Other studies have reported that up to 14% of recovering asymptomatic patients who were tested and negative upon discharge, re-test positive with no sign of infection (31). Also, some studies have reported that recovering patients became symptomatic after a short symptom-free period. A 24-year-old health-worker became symptomatic and tested positive for SARS-CoV-2 within 52 days of an initial symptomatic diagnosis of COVID-19. Similar to our study, no confirmatory RT-PCR testing was performed at discharge. Serum antibodies were not detected at the beginning of the second symptomatic period, which can represent an incomplete immune response that left the infection temporarily dormant, only to be re-activated again; and even though the patient was symptomatic and had had occupational contact with infected patients during her symptom-free recovery period, re-activation was considered more probable than re-infection (32). Similar studies have reported a return of mild or even severe symptoms; but within a short time frame and without genetic analysis of the infective pathogens, they were reported to be cases of reactivation rather than re-infection (33–36).
The second follow-up RT-PCR
Out of the 44 patients who tested negative on the first test and continued their cooperation with the study, 3 patients (6.81%) tested positive 60 days after the initial positive RT-PCR test (one month after the first negative RT-PCR test). These patients again were not symptomatic and the time laps between the initial infection and this positive test is not significant enough to rule out viral shedding. Although, in case of viral shedding and/or presence of remnant viral particles, we would have expected to achieve a positive result in the previous RT-PCR test as well as the second test. Due to the negative result of the first follow-up test, we believe our results can possibly point to re-infection, but this dissonance in results can be explained in absence of re-infection. Given the limited accuracy of RT-PCR, in case of a false negative in the first follow-up test or a false positive in the second test, continued shedding or complete recovery (respectively) can be misdiagnosed as re-infection (37, 38). On the other hand, studies have shown that COVID-19 patients have a lower concentration of ACE2 monocyte expression—the endogenous entry receptor of SARS-CoV-2—and researchers have hypothesized that the virus can remain dormant in peripheral blood mononuclear cells and cause a relapse after the respiratory system has been cleared of the virus and patient has tested negative (39–41).
Presence of typical symptoms, which cannot be explained by any diagnosis other than SARS-CoV-2 infection is also a strong indicator of active infection, which may represent re-infection or re-activation of the initial infection (23). Positive RT-PCR tests in absence of significant symptoms in a recovering patient has also been reported in many studies (18, 42). In some reported cases, symptoms were present, although less severe than the first episode (43). Our findings include asymptomatic and mildly symptomatic patients who re-tested positive for SARS-CoV-2. None of our cases had symptoms as or more severe than the first episode of infection. Contrasting our results, the majority of other reports have described more severe symptoms in patients who re-tested positive (15–17, 44) and researchers have hypothesized that a selection bias towards testing and confirming re-infection in symptomatic patients (45) and/or a primed and heightened immune response upon the second course of infection can be the reason why most cases of re-infection pertain to patients with more intense symptoms upon second exposure and infection (15, 44).
The strongest proof of an episode of re-infection can be achieved by a positive viral culture (23, 46) and/or genetic sequencing of the infective virus in both episodes of infection to confirm that the second episode is caused by a different clade or lineage of the virus. Tillett et al, reported a case of re-infection in a 25-year-old male, who recovered from a RT-PCR-confirmed episode of COVID-19, only to become symptomatic again after a 30-day symptom-free period. Genetic sequencing showed a distinct genetic difference between the two SARS-CoV-2 specimen, indicating two separate instances of infection with genetically different variants of the virus (15). Similar cases of re-infection with a genetically different clade of the virus within 6 months of an original episode of COVID-19 have been reported (16–18).
Unfortunately, genetic sequencing is not readily available or performed and based on Center for Disease Control and Prevention (CDC) recommendations in absence of genetic proof of infection with a different clade of the virus, a positive RT-PCR test that has been obtained after the first 90 days of the onset of the initial infection can be considered indicative of re-infection. Although, a positive RT-PCR test after two consecutive negative results, especially if accompanied by typical symptoms, can be defined as re-infection even within the first 90 days of the first episode of infection (23).
The third follow-up RT-PCR
According to the recommended CDC definitions (30), re-infection is a probable diagnosis for the three symptomatic patients who tested positive in the last RT-PCR screening test; and it is less likely in case of the one asymptomatic patient. The third follow-up RT-PCR screening was performed 120 days after the initial diagnosis of COVID-19 on 32 patients who had tested negative on both of the previous tests. Even though we did not perform viral cultures to prove presence of replication-competent virus, the mild symptoms that these patients experienced can be considered as indicators of re-infection. Although, the symptoms could have been caused by re-activation of dormant infection and release of viruses from body reservoirs. A similar process involving latent infection of cells followed by transcription of viral genome has also been suggested, which would result in reactivation of the virus from a latent to a lytic stage after a symptom-free period, causing a resurgence of COVID-19 symptoms (41); but the long time gap between the two positive RT-PCR results makes re-activation an unlikely diagnosis (23). These four patients were not significantly different from those who tested negative in terms of past medical history or severity of the initial episode of COVID-19. Also, we did not find any specific risk factors that could help us to distinguish patients who are more susceptible to re-infection from those who are not.
The normal human response in COVID-19 is comprised of both humoral and cellular responses and production of CD4 + and CD8 + cells. Both insufficient and overactive immune responses have been reported in these patients (47). Production of protective cytokines and IFN-gamma, which is mediated by the activation of the CD4 + and CD8 + cells, plays an important role in containing and resolution of the infection (48). The dynamics of the antibody response in COVID-19 patients is not completely known; and different studies have reported different rates of seroconversion among these patients. Zhao et al (49) and Liu et al (50) reported seroconversion in all infected patients respectively by 39 and 14 days after the onset of the infection. Liu et al also reported that by the 60th day IgM antibodies were undetectable in about one-third of the patients and the IgG titers had decreased substantially (50). Based on another study, even though recently discharged patients have a high level of humoral immunity against the virus, IgG and neutralizing antibodies start to decreases within 2 to 3 months after the infection (13). In another study the seroconversion rate for IgG, IgM and IgA was ~ 90% and most patients seroreverted within 75 days; although IgG levels remained detectable for over 90 days after the symptom onset in more than 99% of patients (51). Multiple other studies have also concluded that the humoral immunity against this virus could be short-lived (52). Contrasting these studies, our results showed that 94% of patients were positive for neutralizing antibodies (IgG) 120 days after the onset of symptoms. Our results are in line with the results of an Icelandic population study that reported a 91% seropositivity four months after the initial diagnosis of COVID-19 (6). In evaluation of the results of these studies, we should take into account the dynamics and natural process of the humoral response. In case of many other viral infections—where seroconversion is sustained as seromaintenance and immunity—we see a temporary decrease of antibody levels during the first few months of infection/inoculation (53), and since the emergence of COVID-19 is recent, we cannot judge the efficacy of the humoral response to this virus in long term and a rebound increase in antibody levels can be expected (52).
In our study, the four patients who had a positive result in RT-PCR screening 120 days after the initial diagnosis of COVID-19, were also positive for neutralizing antibodies; and although they theoretically may have prevented a severe episode of re-infection and caused a lack of any symptoms in one RT-PCR-positive patient, we cannot know for sure if those levels are high enough to be completely protective in each patient (23). In a similar study Zhang et.al reported re-infection in 6 recovered COVID-19 patients that was caused by viruses from lineages different than the first infection. They reported that all these patients had varied levels of neutralizing antibodies and concluded that presence and even maintenance of the humoral response cannot rule out the possibility of re-infection (54). We also believe that the two patients who did not have sufficient levels of IgG (< 1.1 g/L), have been protected from an episode of re-infection by a strong cellular immune response, even within an epidemic situation.
Genetic sequencing of the respiratory samples, can rule out a false positive in RT-PCR testing and determine if the infection is caused by a different subclass of the virus. In cases of re-infection with a clade of the virus different from the first episode, even protective levels of IgG may not be effective against the new strain of the virus (23). So, we hypothesize that high levels of neutralizing antibodies do not make the diagnosis of re-infection unlikely, unless there is genetic proof that the positive RT-PCR results are related to the same strain of virus that caused the first episode; in which case re-activation/relapse would be a much more likely diagnosis.
One of the limitations of our study was that a considerable number of patients, who participated, dropped out before completion of the study and that this study was only performed in one medical center. We did not perform viral genetic sequencing to prove infection with a different clade of the virus, or viral cultures to prove presence of replication-competent virus as opposed to residual viral fragments in case of viral shedding. The diagnosis of re-infection in our study is exclusively based on RT-PCR results, typical symptoms and the long time gap between the two positive RT-PCR results. And, since we did not measure the viral load in patients who tested positive, the diagnosis of re-infection in our study is as certain as is the specificity of the RT-PCR test; which due to our limited resources could not be repeated 24 hours after each session in order to further avoid false results. We were also unable to perform required serology testing from the beginning of the study, so we cannot investigate the changes of antibody levels throughout the course of the disease.