**1. Introduction**

SARS-CoV-2, a novel enveloped RNA beta coronavirus, has manifested a variety of clinical characteristics from asymptomatic infection to severe pneumonia, vasculitis and death [1–3]. The World Health Organization (WHO) declared this disease a pandemic on 11 March 2020. As of 29 December 2020, SARS-CoV-2 infected more than 81 million people worldwide, with 1,784,533 deaths [4]. Bangladesh officially declared its first COVID-19 case on 8 March 2020 [5]. By 30 December 2020, 512,496 cases and 7531 deaths were reported in the country [6].

Direct, indirect or close contact with infected people and exposure to their saliva and respiratory droplets, which are released when an infected person coughs, sneezes, or speaks, may cause viral transmission [7]. The median incubation period of this virus is about 5 days and the infectious period duration is approximately 10 days [8,9]. A meta-analysis found that 59% of all transmission came from asymptomatic carriers [8].

**Citation:** Das, P.; Satter, S.M.; Ross, A.G.; Abdullah, Z.; Nazneen, A.; Sultana, R.; Rimi, N.A.; Chowdhury, K.; Alam, R.; Parveen, S.; et al. A Case Series Describing the Recurrence of COVID-19 in Patients Who Recovered from Initial Illness in Bangladesh. *Trop. Med. Infect. Dis.* **2021**, *6*, 41. https://doi.org/10.3390/ tropicalmed6020041

Academic Editor: Peter A. Leggat

Received: 26 January 2021 Accepted: 23 March 2021 Published: 31 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

WHO defines a laboratory confirmed case of COVID-19 if the patient's sample (at least a single nasopharyngeal (NP) and/or oropharyngeal swab or wash and/or lower respiratory sputum and/or endotracheal aspirate or bronchoalveolar lavage) tests positive by real-time reverse transcription-polymerase chain reaction (rRT-PCR) [10]. rRT-PCR assays only inform clinicians whether SARS-CoV-2 is present or not. However, these assays also provide quantitative data on cycle threshold (Ct) values, which are inversely related to the viral load and are not reported clinically. An advanced assay (e.g., whole genome sequencing) is required to interpret a recurrence—whether it is a relapse or a true reinfection. The effect of SARS-CoV-2 viral load on clinical outcomes and recurrence has not been extensively studied.

Generally, "relapse" may be defined as a "recurrence" with the same species and strain of a micro-organism that was present before, whereas "reinfection" is a secondary infection with a different species or strain. Reinfections with respiratory viruses may occur as a result of a weakened or waning immune response (e.g., respiratory syncytial virus), reinfection with a different genotype/species (e.g., rhinoviruses), or the viruses' high variability (e.g., influenza virus). The immunity against SARS-CoV-2 is not well established and it is uncertain how long the antibody will prevent re-infection [11]. Moreover, vaccineinduced protective immunity may differ from natural immunity due to the immuneevasion strategies of wild-type viruses [12]. However, there is some evidence of persistence of neutralizing antibodies in COVID-19 patients with natural infection for the first few months (~8 months) [13,14]. The immune response following a natural infection is thought to be incomplete, and reinfections are likely [15]. One should not confuse relapse with "long COVID" which refers to symptoms of COVID-19 that continue after the typical convalescence period. According to some reports, about 10% of people who tested positive for SARS-CoV-2 had one or more symptoms for more than 12 weeks [16].

Reinfection with SARS-CoV-2 is rare but possible, according to recent studies. Positive RT-PCR results in patients who recovered from initial illness have been reported in a number of countries [17]. In August 2020, a reinfection case was confirmed in Hong Kong in a patient with clearly different genome sequences [18]. Three confirmed reinfection cases were also reported from Nevada, USA [19], Belgium [20] and Ecuador [21].

There are some anecdotal reports of reinfection/relapse from Bangladesh. Among the positive COVID-19 staff cases from the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), the investigators noticed that some patients who clinically recovered from their illness (RT-PCR negative) were found to be RT-PCR positive for SARS-CoV-2 a second time. The aim of our study was to investigate suspected cases of reinfection or relapse excluding long COVID. We explored the clinico-epidemiological data of recurrent COVID-19 cases in Bangladesh and correlated these findings with their RT-PCR test results and whole genome sequencing.

#### **2. Materials and Methods**

#### *2.1. Study Design and Setting*

We conducted a case series of icddr, b staff between March and September 2020 where the community transmission of SARS-COV-2 was established.

#### *2.2. Study Population and Procedure*

The International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) is a leading public health research institution in South Asia with approximately 4000 staff. Since 21 March 2020, all staff with clinical features (fever, cough, cold or respiratory distress) of COVID-19 were instructed to contact the icddr,b Staff Clinic. The staff clinic doctors advised any suspected staff member to undertake a nasopharyngeal swab for a COVID-19 test using RT-PCR. Some high-risk staff, such as staff nurses collecting NP swabs and laboratory personnel working with SARS-CoV-2, were also advised to undertake routine COVID-19 tests.

When a patient was RT-PCR positive, staff clinic doctors advised patients for home isolation if the condition was not severe enough for hospitalization. The staff clinic also prescribed and distributed medicines for each COVID-19 positive patient. Along with severe cases, less severe cases were also admitted to the isolation center of icddr,b if the staff member had difficulty with home isolation. All positive cases were advised to have a repeat test on day 14 and every week thereafter until tested negative.

The WHO criteria [22] for withdrawing from isolation for symptomatic patients as: 10 days after symptom onset, plus at least three additional days without symptoms (including fever and respiratory symptoms); and for asymptomatic cases: 10 days after a positive test for SARS-CoV-2. Based on the WHO case criteria and recent publications [23], we set the following criteria for selecting patients to investigate for reinfection: (1) an initial SARS-CoV-2 PCR-confirmed diagnosis; (2) followed by clinical recovery and with at least one negative SARS-CoV-2 PCR result; (3) followed by a confirmed SARS-CoV-2 PCR positive result from a single nasopharyngeal swab test with clinical symptoms at least 28 days after the previous SARS-COV-2 negative PCR result. The WHO clinical progression scale [24] specific for COVID-19 was used for clinical classification and assessing disease severity.

### *2.3. Laboratory Investigations*

All participants submitted a single nasopharyngeal swab that was collected by trained nurses in viral transportation media. The specimens were transported to the laboratory in coolers within an hour of collection. RNA was extracted from the nasopharyngeal samples using QiaAmp Viral RNA Mini kit (Qiagen, Hilden, Germany). A final volume of 60 microliters of RNA was eluted from a 140-microliter sample. RNA was tested for SARS-CoV-2 by real-time reverse transcription polymerase chain reaction (rRT-PCR) targeting ORF1ab- and N-gene specific primers and probes following the protocol of Chinese Center for Disease Control and Prevention (briefly as China CDC). A positive case was determined if the CT values of two targets (ORF1ab and N) were <37 in the same specimen. If CT values of any sample were 37 −40 or a single target was positive, it was resampled and retested. If the CT values were still 37 −40 and the amplification curves had obvious peaks, the sample was considered positive. We did repeat tests for all second-time positives to confirm that they were not false-positive cases. If a single target was positive in the repeat test, it was determined to be positive. The complete sequencing of positive isolates from the first and second episodes of each case was conducted using the Illumina NextSeq 500 platform. The RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA Gold Library kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. The normalized pooled library was sequenced employing the Illumina NextSeq v2.5 sequencing kit (Illumina, San Diego, CA, USA). Additionally, all samples were tested for common respiratory viruses including: influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza (1, 2, 3), human metapneumovirus (hMPV), and adenovirus using real-time PCR following standard procedure [25,26].
