Recurrence of Positive SARS-CoV-2 RNA in a COVID-19 Patient: Two Case Reports from Saudi Arabia

Background The rapid spread of the COVID-19 pandemic which took a place in Wuhan, China and diffused globally required the international public health to take a coordinated international response. As other coronaviruses were described, SARS-CoV-2 was thought to be an immunizing, monophasic disease. Herein, we reported rst two COVID-19 cases registered in Saudi Arabia whose nasopharyngeal swabs turned positive for SARS-CoV-2 ribonucleic acid (RNA) after recovery. Case presentation First patient who is symptomatic with recurrence of positive SARS-CoV-2 RNA occurred 115 days after the rst symptomatic infection and did not develop protective immune response. Second patient who is asymptomatic during the recurrence of positive SARS-CoV-2 RNA occurred 60 days after the rst symptomatic infection in an apparently immunocompetent patient. Conclusion These ndings highlight the recurrence of positive SARS-CoV-2 after recovery even if SARS-CoV-2 antibodies were developed.


Background
Since the fast expansion of the ongoing global pandemic Severe Acute Respiratory Syndrome  in December 2019, the number of Coronavirus disease (COVID-19) cases have exceeded 27 million worldwide [1]. Saudi Arabia was among the impacted countries that reported the rst case of COVID-19 on March 2, 2020. By September 8, 2020, the Ministry of Health (MOH) reported over 322,000 positive cases, with 4137 deaths and 298,246 recoveries [2].
COVID-19 is a respiratory illness, person to person contact, and respiratory droplets are the primary route of infection. When the pandemic rst started, it was thought to be an immunizing, non-relapsing disease [3]. However, in some countries, there have been reports of rare suspected cases of COVID-19 reinfection [4]. Herein, we reported two COVID-19 cases registered in Saudi Arabia with recurrence of positive SARS-CoV-2 ribonucleic acid (RNA).

Case Presentation
First case Patient 1 is a 23-year old healthy male, with no underlying medical conditions, who had a nasopharyngeal (NP) swab collected on April 13 using a viral transport media (VTM). As recommended by the Saudi Ministry of Health, the diagnostic testing for SARS-CoV-2 was performed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using at least one con rmatory target, in addition to the screening targets [5].The RT-PCR test was done as a standard infection control procedure after the patient arrived from a trip abroad. The test result was positive and con rmed by the National Health Laboratory (NHL; Table 1). Two days later, the patient started complaining from joints pain, mild headache, and diarrhea. According to the Saudi Ministry of Health guidelines for COVID-19 positive patients, the local Page 3/8 health authorities urged him to self-isolate until his symptoms resolve, and the subsequent test is negative [6]. He received no prescription medications during his quarantine period. To follow up, on April 20 and 25 two NP swabs were collected for RT-PCR assay test at two different laboratories, and both results were negative. As a result, the patient resumed his normal life. Just over three months later (August 6), he developed the following symptoms: moderate headache, fatigue, sore throat, and diarrhea. He suspected reinfection due to close contact with COVID19 patient and performed two SARS-CoV-2 RT-PCR tests at two different laboratories; both were positive. The symptoms were subjectively more intense this time and lasted for three days. A follow-up and con rmatory tests were carried out on August 13 and 16, respectively, and both yielded negative results. For further investigations, a SARS-CoV-2 IgG assay was conducted (August 16) for the detection of SARS-CoV-2 antibodies development, and it came out negative with a titer of 0.16 AU/mL (Reference value < 0.7 AU/mL) indicating a possible non-protective immune response [7].  (Table 1). A week later, the patient's health started to improve, and symptoms began to disappear gradually. On July 1 and 4, two consecutive follow-up tests were done, and both results were negative for SARS-CoV-2, which suggested that the patient was cured. At the beginning of August, some of the patient's family members were infected with SARS-CoV-2 experienced u-like symptoms such as fever, fatigue, and headache. Although the patient himself did not experience any symptoms, he decided to do a cautionary SARS-CoV-2 test on mid-August, and it revealed a positive result which was also con rmed on a subsequent NP swab collection. Furthermore, we performed IgG assay at that time, which came back positive (titer of 5.7 AU/mL), con rming the presence of SARS-CoV-2 antibodies.

Discussion And Conclusion
We report here two cases of healthy adults who had two episodes of possible COVID-19 infection. Interestingly, in one case, the symptomatic patient with recurrence of positive SARS-CoV-2 occurred after three months of the initial infection that did not trigger a protective immune response. In the other case, asymptomatic patient with recurrence of positive SARS-CoV-2 occurred within three months of the initial infection.
Recently, Yao et al. demonstrated that SARS-CoV-2 viral particles remain in previously positive patients' lungs whose NP swabs results were negative three times consecutively; this suggests that SARS-CoV-2 virus may not be eliminated when the symptoms disappear [8]. Therefore, there is still a reason to be cautious, and we need to be aware of recovered patients and their potential infectivity. Interestingly, there have not been any studies demonstrating that recovered patients are infectious.
To our knowledge, our cases are the rst to describe positive SARS-CoV-2 viral RNA in recovered COVID-19 patients in the Middle East. According to the Centers for Disease Control and Prevention (CDC), reinfection of SARS-CoV-2 is highly considered in a patient who becomes symptomatic around 90 days after onset of infection and has been in close contact with an infected person, supporting our rst case [9]. A similar case presenting recurrence of SARS-CoV-2 has been reported in Hong Kong, bringing more attention lately. To and his colleagues demonstrated that second episode of asymptomatic infection occurred after the rst symptomatic episode in an apparently immunocompetent patient [4]. In addition, they con rmed SARS-CoV-2 reinfection by performing whole genome sequencing for the rst and second episodes indicating different clades [4]. We assume that the recurrence of COVID-19 is possible due to the presence of nonprotective IgG and recovered patients might be carriers and actively transmitting the virus even after their two negative tests [10,11]. Thus, gatherings of positive cases are not safe, and there should be an emphasis upon such social practices, and for those in convalescence, we suggest that they undergo regular tests for infectivity assessments.
The presence of SARS-CoV-2 virus appears to be unstable because of the possibility of false-negative results of the molecular test which might be due to uctuations in the viral load or pre-analytical errors during the sample collection [12]. In our case reports, reducing the chance of false-negative and falsepositive results was done by con rming each test result in different laboratory with new NP swab collection. For both cases, rst positive test results were con rmed by the NHL, and two consecutive negative tests were done before the patients were considered to be cured. Although these are only two case reports and the evidence is not determinant, it gives insight and encourages future cohort studies.
In conclusion, the global public health is at risk of the possibility of recurrence of SARS-CoV-2 as we exhibited in our case reports, which could contribute to the spread of the virus. Therefore, additional virologic, immunological, and epidemiological studies should be done to have better understanding of SARS-CoV-2 recurrence mechanism.

Consent of publication
Written consent was obtained from the patient for publication.

Availability of data and material
Derived data are available on reasonable request.
Competing interests