Distinguishing repeated polymerase chain reaction positivity from re‐infections in COVID‐19

Abstract Background Possibility of reinfection with SARS‐CoV‐2 changes our view on herd immunity and vaccination and can impact worldwide quarantine policies. We performed real‐time polymerase chain reaction (RT‐PCR) follow‐up studies on recovered patients to assess possible development of reinfections and re‐positivity. Methods During a 6‐month period, 202 PCR‐confirmed recovering COVID‐19 patients entered this study. Follow‐up RT‐PCR tests and symptom assessment were performed 1 month after the initial positive results. Patients who tested negative were tested again 1 and 3 months later. The serum IgG and IgM levels were measured in the last follow‐up session. Results In the first two follow‐up sessions, 82 patients continued their participation, of which four patients tested positive. In the second follow‐up 44 patients participated, three of whom tested positive. None of the patients who tested positive in the first and second follow‐up session were symptomatic. In the last session, 32 patients were tested and four patients were positive, three of them were mildly symptomatic and all of them were positive for IgG. Conclusions A positive RT‐PCR in a recovering patient may represent reinfection. While we did not have the resources to prove reinfection by genetic sequencing of the infective viruses, we believe presence of mild symptoms in the three patients who tested positive over 100 days after becoming asymptomatic, can be diagnosed as reinfection. The immune response developed during the first episode of infection (e.g., IgG or T‐cell mediated responses that were not measured in our study) may have abated the symptoms of the reinfection, without providing complete protection.

the first and second follow-up session were symptomatic. In the last session, 32 patients were tested and four patients were positive, three of them were mildly symptomatic and all of them were positive for IgG.
Conclusions: A positive RT-PCR in a recovering patient may represent reinfection.
While we did not have the resources to prove reinfection by genetic sequencing of the infective viruses, we believe presence of mild symptoms in the three patients who tested positive over 100 days after becoming asymptomatic, can be diagnosed as reinfection. The immune response developed during the first episode of infection (e.g., IgG or T-cell mediated responses that were not measured in our study) may have abated the symptoms of the reinfection, without providing complete protection. Coronavirus 2 (SARS-CoV-2) has infected more than 134 million people worldwide and has caused more than 2 million deaths. 1 No curative drug or specific treatment is known to have considerable efficacy against this infection; but, currently, over 200 candidate vaccines have been acknowledged by the WHO. [2][3][4] Our experience with other respiratory infections, such as those caused by influenza and seasonal respiratory infections caused by coronavirus lead us to believe that the natural immunity after an episode of infection with this virus may not cause long-time immunity. 5 Some studies have shown a substantial wane in antibody levels within a few months after remission 6,7 ; and the studies that report re-infection further question the efficacy and longevity of the acquired immunity after infection with this virus. Several cases of suspected and proven instances of re-infection with SARS-CoV-2 have been reported in immunocompetent patients in different age groups, in patients with different levels of antibody response. [8][9][10] The duration of sustained antibody response after an episode of infection is generally a representative of the immune protection that can be achieved by vaccination against the same virus. 11 Waning of the IgG antibodies that are naturally produced in a patient after infection with SARS-CoV-2, undermines the supposed efficacy of vaccination; especially since cases of symptomatic re-infection with replication-competent virus have been reported within the first 6 months after the initial infection. 12,13 To this date, thousands of distinct variants of SARS-CoV-2 have been identified with over 400 variants in the spike protein, which is presumably the binding site of neutralizing antibodies. 14 Besides, in many cases of re-infection, genetic sequencing has revealed a different clade of the virus to be the causative pathogen. [15][16][17][18] We can assume-as is the case with infections caused by rhinoviruses and influenza-that the protective activity of antibodies is limited to each specific subtype of the virus, this can possibly explain why re-infection can occur in presence of detectable levels of IgG. 19,20 In this study we investigated the rate of symptomatic and asymptomatic re-positivity with SARS-CoV-2 in recovering patients for up to 4 months after the initial diagnosis of Coronavirus Disease 2019 (COVID-19); and find possible risk factors that are associated with reinfection.

| Eligibility criteria
Patients who had been diagnosed with COVID-19 (approved by a real-time polymerase chain reaction [RT-PCR] test of nasopharyngeal specimens) and had been admitted based on the national criteria for hospitalization (a sustained peripheral oxygen saturation of under 93% and/or a respiratory rate of over 30/min or sustained nausea and vomiting and severe weakness even with normal oxygen saturation), were brought into the study upon discharge. The current national discharge criteria dictate that prior to discharge patients must have at least two consecutive afebrile days with a blood oxygen saturation of over 90%, oral intake without nausea, and improvement in weakness. The exclusion criteria were a negative RT-PCR result or lack of documentation of a positive result at the initial hospitalization.

| Study initiation and follow-up sessions
All information regarding patients' admission and epidemiological data including age, sex, any past medical history, and recent use of immunocompromising medications were documented.
The first follow-up RT-PCR study was performed 1 month after the initial positive RT-PCR test; at which point all patients had been asymptomatic for at least 14 days. All patients who tested negative were re-tested 1 and 3 months after the date of the first follow-up RT-PCR test (Figure 1). A complete assessment of signs and symptoms related to COVID-19 along with serology testing for anti-SARS-CoV-2 IgG and IgM levels were also performed during the third follow-up visit.

| Antibodies
A 5 cc whole blood sample was drawn (without anticoagulants), and serum was derived from the specimen using centrifugation (3000xg for 10 min). The Enzyme-linked Immunosorbent Assay (ELISA) method (Pishtaz Teb SARS-CoV-2 IgM and IgG Iran) was used to test the serum antibody levels. The test was performed according to the manufacturer's brochure. Results greater than 1.1 were considered positive and those less than 0.9 as negative. Results within the mentioned range were reported as borderline and the test was redone on a second, fresh serum sample to confirm the initial results.

| Measurements and statistical analysis
Data were analyzed using SPSS software V.22.0. Quantitative variables are reported by mean and standard deviation (SD) and qualitative variables are reported using frequency and percentage.
Chi-square and Fisher's exact tests were used to assess the statistical relationships between categorical variables. The level of significance was set as P value < .05 for all analyses.

| Ethical considerations
Informed consent was obtained from participants and they were advised that they can leave the study at any time point and this will not hinder their current treatment or future visits to the hospital and the quality of care they would receive. This study was conducted in  Patients had a median age of 47 ranging between 29 and 84 years old. Table 1 shows the demographic description of participants. The first RT-PCR results of four patients (4.87%) were positive; who were asymptomatic and not different from those who tested negative in terms of the duration of the initial admission, the severity of the first episode of the disease, presence and type of underlying diseases, and recent history of using immunosuppressive drugs (P value: .63, .57, .59, and .61).   In some studies, a positive RT-PCR in a recovering patient who tested negative upon resolution of their symptoms has been considered a strong indicator of. 10 Although, false results are possible. A false positive result can occur in a recovering patient; also a false negative result shortly after subsidence of symptoms, followed by a correctly positive test misleads physicians toward a re-infection diagnosis. [22][23][24] 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. 25,26 Also, RT-PCR cannot differentiate replication-competent viruses from viral fragments that are expelled from a recovering patient. 27,28 Viral shedding from the respiratory tract during recovery has been reported to last for as long as 12 weeks after infection 29 ; thus, a significant time-gap between the first episode of infection and a positive RT-PCR can clarify that the patient has passed the viral-shedding stage.

|
In this study RT-PCR testing was not performed upon discharge to confirm viral clearance. The first follow-up RT-PCR study was per-  Chronic pulmonary dis. after the respiratory system has been cleared of the virus and patient has tested negative. [38][39][40] Positive RT-PCR tests in absence of significant symptoms in recovering patients have also been reported in many studies. 18,41 In some cases, symptoms were present, although less severe than the first episode. 42 Contrasting our results, the majority of other reports have described more severe symptoms in patients who re-tested positive [15][16][17]43  but the long interval between the two positive RT-PCR results makes re-activation an unlikely diagnosis. 21 We did not find any risk factors that could help distinguish patients who are more susceptible to reinfection from those who are not.

| Humoral response
Both insufficient and overactive immune responses have been reported in COVID-19 patients. 46 The dynamics of the antibody response in COVID-19 patients is not completely understood; and different rates of seroconversion have been reported. Zhao et al. 47 and Liu et al. 48 reported seroconversion in all infected patients respectively by 39 and 14 days after the onset of 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. 48 Another study showed recently discharged patients have an even high levels of antibodies start to decrease 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; with IgG levels remaining detectable over 90 days after the symptom onset in more than 99% of patients. 49 Multiple studies have also concluded that the humoral immunity against this virus could be short-lived. 50 Contrasting these studies, our results showed that 94% of patients were positive for neutralizing antibodies (IgG) 120 days after the onset of symptoms; which is in line with the results of an Icelandic population study that reported a 91% seropositivity 4 months after the initial diagnosis of COVID-19. 6 To evaluate these results, we should take into account the 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, 51 and since the emergence of COVID-19 is recent, we could expect a rebound increase in antibody levels later on. 50 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 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. 21 In a similar study Zhang et.al reported re-infection in six recovered patients that was caused by viruses from lineages different from the first infection. All these patients had varied levels of antibodies and they concluded that presence and even maintenance of the humoral response cannot rule out the possibility of re-infection. 52 We 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.
In cases of re-infection with a different clade of the virus, even protective levels of IgG may not be effective. 21 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 the virus from the first episode; in which case re-activation/relapse would be a more likely diagnosis.

| Limitations
Some of the limitations of our study were that a considerable number of participants dropped out during the study, this study was only performed in one medical center, and that we could not perform genetic sequencing to prove infection with a different clade of the virus, or cultures to prove presence of replication-competent virus. The diagnosis of re-infection in our study is based on RT-PCR results, typical symptoms and the long interval between the two positive 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. We were also unable to perform required serology testing from the beginning of the study.

CONFLICT OF INTEREST
None.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.12883.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical restrictions.