Detection of SARS CoV-2 reinfections by rapid inexpensive techniques

Reinfections of SARS CoV-2 are rare in the world and it is dicult to be conrmed whether it is a reinfection or persistent infection. The most prominent factors used for differentiating the reinfections from persistent infections are whole genome sequencings and phylogenetic analyses that require times and funds, which may not be feasible in most developing countries. We previously developed rapid economical methods to identify both D614G and N501Y mutations in clinical samples using rRT PCR probes and endpoint PCR specic primers. Current study has found an immunocompetent patient with a SARS CoV-2 N501Y reinfection without comorbidities. The results suggested that the initial infection was due to a variant contained only D614G mutation while the reinfection was potentially as result of the UK variant contained three mutations conrmed by DNA sequences, including D614G, N501Y and A570D mutations. Seven cases of reinfections were also conrmed by these methods suggested that these techniques will support rapid detection of SARS CoV-2 reinfections in developing countries where sequencing tools are unavailable.

Introduction SARS CoV-2 reinfections are rare and happen as a result of declining antibodies in convalescent people.
However, recent study found that a reinfection occurs in an immunocompetent person who had neutralizing antibodies produced from the initial infections (1). The infections generally persist in immunosuppressed patients but the reinfections occur in immunocompetent individuals in regions where different variants are circulating (2); this is often confusing to differentiate between reinfections and persistent infections. The reinfections can be differentiated from persistent infections by identi cations of the viral variants in the clinical samples taken from both rst and second infections. Viral variants are usually identi ed by phylogenetic analysis of the viral whole genome sequences which are di cult to obtain as quickly as possible for both initial and second infections (3). In other words, con rmations of reinfections have been under investigations. CDC recommends several criteria for suspecting SARS CoV-2 reinfections including collecting respiratory samples daily for 7 days, and serum samples in different times points in days 3, 7, 14, 21, 60 (4). Therefore, alternative, rapid, molecular biological methods can also be exploited for variant identi cations using rRT PCR probes, particularly when whole genome sequencings are not available in poor or developing countries. Several methods have recently been developed (5)(6)(7)(8)(9). These methods are more inexpensive and feasible than whole genome sequencings.
Before emergence of SARS CoV-2 variants of concerns such as B.1.1.7, B.1.351, P1 and B.1.617, reinfections were seldomly reported in the world. For example, a reinfection with SARS CoV-2 was rst reported in a person from Hong Kong migrated to Europe that was con rmed to be caused by a different lineage of the virus (10). Later on, several cases of re-infections have been reported worldwide (2).
However, the variants of most of these reinfections were not con rmed by genomic sequencings; this makes reporting reinfections controversial and confusing with persistent infections (11). In other words, the majority of the global SARS CoV-2 reinfections may have been a persistent infection not even reinfections (12). This highlights the importance of the reinfection con rmation and persistent infection exclusions by using either whole genome sequencings or rapid techniques to identify the virus's variants.
SARS CoV-2 has three major variants of concerns (VOC), which have been known to in uence on the transmissibility, infectivity and fatality of the virus, including B.  (15,16). Nonetheless, no studies have con rmed reinfection using DNA sequencings, particularly with the UK variant and few reports in the world. This was possibly due to di culties in comparing the whole genome sequencings between the initial infections and reinfections. In this study we aim to investigate reinfections in a person reported in Kalar town, Sulaymaniyah province, Kurdistan regional government of Iraq, where sequencing facilities are hardly obtained. We also aim to apply the previous rapid methods to con rm reinfections with variants carrying N501Y mutations that have occurred in the region.

Materials And Methods
Firstly, a person was presented to Coronavirus Research and Identi cation lab, University of Garmian, Kurdistan Region, Iraq. He agreed to ll a consent form as a participant of this study, which was approved by an ethical committee at the Department of Biology, University of Garmian that follows the rules adhered to the Declaration of Helsinki for human and animal research. He was 42 years old, his body weight was 70, height= 170 cm. Nasopharyngeal swab was taken in viral transport medium (VTM) on 7 th October 2020. The initial infection was tested for diagnosis as SARS CoV-2 using a coronavirus real time RT-PCR kit (MutaPLEX® immundiagnostik, Germany). Blood tests were performed to observe complete blood counts (CBC), ferritin, D dimer, LDH, CRP and ESR. The patient had no comorbidities. The reinfection was diagnosed on 23 March 2021 (after 5.5 months) using same protocol as the initial infection.
Identi cations of SARS CoV-2 N501Y and D614G mutations were performed for both initial infection and the reinfection, using rapid molecular biological methods as previously developed (5, 6). These were con rmed by DNA sequencings. An ELISA test for SARS CoV 2 IgG and IgM (Ideal Tashkhis Atieh co. Iran) was performed a day post re-infection. The rapid molecular methods were also applied to 7 persons suspected with either reinfections or persistent infections. They agreed to ll a consent forms and approved by the ethical committee as previously mentioned.

Initial infection
The clinical features were as follows: severe sore throat with excessive cough for two weeks that was initially dry but wet and purulent at the end. Sneezing, malaise, fatigue, diarrhoea were also present. Oxygenation saturations was normal as indicated by normal SPO2 more than 96%. The results of the rapid methods showed that the initial infection resulted from a SARS CoV-2 variant, which have had N501 wildtype and D614G mutant. The viral load was Ct value= 17.14. The sequencing result also confirmed amino acids N501 wildtype, A570 wildtype, D614G mutant in the initial variant (MW897351). Treatment was Vitamin D, Zinc, Vitamin C, Paracetamol and Azithromycin for 10 days. PCR result was negative after 2 weeks. Another PCR test was performed 4 months later.

Reinfection
The clinical features were as follows: moderate sore throat with mild coughs for 10 days. Dry mouth was much more than the initial infection. Malaise and fatigue were less prominent than the previous infection, and the patient had no diarrhoea. He had normal SPO2. The rapid methods and sequencing result (MW897356) found two amino acid changes N501Y, and A570D, in addition to the D614G which was also present in the initial infection. Presence of Y510, D570 and G614 mutations in the reinfection suggested the UK variant. Other parameters are as follows: Ct value= 17.9, normal CBC, ferritin, D dimer, LDH and ESR, but CRP was 15 mg/dl (normal CRP titer less than 5 mg/dl). Treatment was Vitamin D, Zinc, Vitamin C, Paracetamol and Azithromycin for 7 days. The patient recovered with negative PCR after 2 weeks with no clinical signs. Both IgG and IgM were considered as negative results with very low titres, 0.28 and 0.182, respectively.

Application of the rapid molecular biological methods on other re-infections
Seven persons, who were previously positive for SARS CoV-2 from June to September 2020 where no N501Y mutations were present in the region, were tested for reinfections by the same molecular methods as previously mentioned. Results of the rapid methods showed that all the 7 persons were re-infected with a variant having both D614G and N501Y mutations. The patients information and results of reinfections are shown in Table 1. The results revealed that one out of 7 reinfections carried the wildtype N501 variant (Case No.2); these were confirmed by DNA sequencings. While other 6 persons were re-infected with N501Y mutated variants which are present in the UK, South Africa and Brazil variants.

Discussion
This study has reported a SARS CoV-2 reinfection using rapid inexpensive techniques that can used to make differences between reinfections and persistent infections. The rst case reported in this study was con rmed by the rapid tests and DNA sequencings in Kurdistan Region of Iraq. In addition, theses rapid methods, which can detect both D614G and N501Y mutations, were applied to 7 clinical samples. Out of 7 persons, one was suspected to be either re-infected or suffered from persistent infections because she carried only D614G mutations in both initial and second infections as con rmed by DNA sequencings too. However, she seemed to be re-infected rather than persistent infection as the time between both infections were more than 6 months. The other 6 individuals carried both D614G and N501Y mutations; the latter was not present in the rst infection that suggested reinfections. The re-infected patients includes different ages with both comorbidities and no comorbidities suggested that reinfections with the UK variant may occur in various ages without comorbidities.
SARS CoV-2 reinfections were previously rare in the world as in a study conducted in approximately 9 thousands of positive samples in USA from December 2019-November 2020, only 63 samples (0.7%) were reported as reinfections, which were linked with low antibody responses in the initial infection (17). However, recent research has reported 58 out of 1300 suspected reinfections among about 700,000 positive individuals in India (18). Likewise, 138 out of 28,875 positive cases were reported as reinfections in Denmark (19). An ecological research conducted in the UK concluded no evidence of increasing rate of reinfections with the UK variant (b.1.1.17) which were con rmed by whole genome sequencings (20). During writing of this manuscript, several cases of SARS CoV-2 reinfections were reported in USA, Italy, Columbia, Brazil and Luxemburg, in which their second infections were due to B. For how long antibodies persisted in previously infected persons is uncertain. However, studies suggested that antibodies may remain for approximately 6 months (3). The persistence of antibodies varies from an individual to another or it depends on the severity of the disease or the type of the variants. The antibody status of the rst case was performed a day of the reinfection indicated that his adaptive immunity had no response yet and results showed that both IgG and IgM were negative. This suggested that the reinfected person has had no protective antibodies to prevent the reinfection. Limitations of the current study were lack of checking antibodies in all cases. Despite the small number of samples, this study reported reinfections with SARS CoV-2 N501Y mutant.

Conclusions
This research explores SARS CoV-2 reinfections using rapid low cost methods and reported rst reinfections with a SARS CoV-2 N501Y mutant variant in Kurdistan region of Iraq. Further study is required to apply these methods in large number of samples. This will open our understandings towards the epidemiology and reinfections of the virus.