A 53-year-old woman (Case A) was admitted to the emergency room in our hospital (Gregorio Marañón, Madrid, Spain) on April 3, 2020 due to dyspnoea, fever, cough with expectoration of 24 hours of evolution, and history of bronchial asthma. Blood tests and chest X-ray were ordered and results showed no outstanding changes. The SARS-CoV2 PCR on nasopharyngeal exudate was positive (Ct value 30). Since the patient had no respiratory failure nor other serious conditions, she was discharged from hospital the same day and given symptomatic treatment. The patient remained symptomatic for over two weeks (mainly dyspnoea and fever) with eventual resolution of symptoms. On April 18, a second RT-PCR was performed with a positive result. One month after discharge, the SARS-CoV 2 RT-PCR was repeated (May 12) and this time the result was negative. COVID-19 serology was not available at the time.
The source of infection was undetermined. Case A reported having been confined to her home with her husband during the three weeks prior to the beginning of symptoms. She denied contact with anyone else during confinement. Her husband did not have any symptoms and therefore no RT-PCR was performed.
On August 14, four months and a half (140 days) from her first positive RT-PCR, Case A began to have fever, dyspnoea, cough, and arthromyalgia. Another SARS-CoV2 RT-PCR was performed (August 21) with a positive result (Ct value 22).
On August 25, Case A went back to the emergency room, this time with respiratory failure and multiple bilateral pulmonary infiltrates and was admitted to the hospital. No SARS-Cov-2 antibodies were detected in the admission tests. Upon admission, the presence of lymphopenia, mild hypertransaminasemia, and elevated LDH and CRP stood out.
During the first 48 hours, Case A showed radiological worsening and respiratory failure, exhibiting significant bronchospasm. She was given corticosteroids, remdesivir, and lopinavir/ritonavir. Three RT-PCRs were performed during hospitalization (August 28, and September 2 and 6, all positive (Ct values 21, 33, and 33 respectively). The patient progressively improved, and 17 days after admission was discharged with oxygen therapy. Prior discharge, the COVID-19 serology (SARS-CoV-2 IgG Architect, Abbott, Chicago USA) was repeated (8/9). The result was positive with titers of 7.04.
Epidemiological events preceding reinfection
Twelve days before (August 7) Case A´s second episode, she had had close contact with an uncle (without wearing facemasks, tight physical contact, including kisses and hugs, Figure 1). Besides Case A, no other member in the family participated in that contact with the uncle. Two days later, (August 9) the uncle developed a cough, arthromyalgias, asthenia, and dysthermia. The RT-PCR-SARS-CoV-2 test performed on August 12 was positive (Ct value 19). His symptoms resolved on August 18.
Case A was interviewed to obtain more details from her and her uncle´s epidemiological context. Three of her uncle´s friends had also fallen ill with COVID-19; all four attended the same mosque every Friday and had had additional contacts. One of them was interviewed and referred that the last time all four coincided was at the mosque for the Friday ceremony on August 7. The three friends had subsequent positive RT-PCRs (August 11, 15 and 16; Figure 1). Case A, her husband and daughter had not attended that mosque or met the uncle´s friends.
Epidemiological events following reinfection
On August 25, eleven days after the onset of Patient Case A´s symptoms in the second episode, her husband started to report fever and malaise. He tested positive for SARS-CoV2 PCR (Ct value 25). On August 28 his dyspnoea worsened and an interstitial infiltrate was observed in the upper left lobe. Home isolation was indicated for 14 days.
On August 18 Case A´s daughter, who visited her daily, started with a cough odynophagia, asthenia, and fever of 39 º C. On August 21, she had a positive RT-PCR. Home isolation ended after 14 days, without an RT-PCR control. The daughter´s husband began having symptoms the same day (August 18) and had a positive RT-PCR on August 21 and a second one on September 2. The RT-PCR tests performed on their four children were all positive, but only one of them developed symptoms (starting on August 22) (Figure 1).
Genomic analysis
We first confirmed that the specimens isolated from Case A, who had had positive SARS-CoV-2 RT-PCR results in April (first episode) and August (second episode) belonged to the same patient, as indicated by the identical microsatellite STR-PCR patterns obtained from the human DNA in the corresponding samples (Supplementary Figure 1).
Next, we performed comparative analyses of the SARS-CoV-2 sequences of the strains isolated from Case A in the first and second episodes. The analysis of Episode 1 specimen allowed us to confirm that sequences corresponded to SARS-CoV-2, but it did not offer enough coverage (only 18% of the chromosome offered al least 30X coverage) to determine the complete consensus sequence and call SNPs with high confidence. WGS analysis of Episode 2 specimen provided good coverage (99% of the genome with >30X coverage depth and a total of 790545 mapped reads) and allowed us to determine 16 SNPs relative to the Wuhan-1 reference (7 of them missense variants) (Figure 2) and assign the lineage of the strain (20A).
Next, we decided to extend the genomic analysis beyond Case A, to include i) the strain from the potential source of Case A´s second episode, her uncle, and ii) the strains from two of the uncle´s friends, representing his exposure context. In addition, we included Case A´s husband strain as potential subsequent receptor from Case A. No samples were available for sequencing from Case A´s daughter, her son-in-law, or her granddaughter. The four specimens yielded sequences of enough quality (>93-99% of the genome with >30X coverage depth and 152354-525291 mapped reads; and in one specimen 77.53% and 290438) to allow comparisons throughout the complete genome. Case A and her husband presented identical sequences and both differed from those of case A’s uncle and his friends in 1 SNP (Figure 2). These data indicates that the strain involved in Case A´s reinfection was circulating in the epidemiological context of Case A´s uncle. The acquisition of one SNP, the presence of this SNP shared by Case A and her husband, the chronology of the cases, and the fact that Case A husband did not attend the mosque or met Case A´s uncle nor his friend, suggest a direction of transmission from the uncle to Case A and then from Case A to her husband.
An extended phylogenetic analysis, including 348 strains sequenced in our institution from the same population in Madrid, showed that this strain was circulating by the time Case A suffered the second episode, August/September 2020. The strain was part of a clade including exclusively strains after June 2020 (blue clade) and the five cases in this study shared a single proper branch within this clade (Figure 3). The strain, or related strains, were not found among the strains circulating in the same population in Madrid at the time range corresponding to Case A´s first episode (end of March/beginning of April; red clades; Figure 3). In agreement, a dating analysis available in nextstrain.org shows that the second episode strain belongs to a clade, 20A.EU1, that had no representative sample before the end of June 2020 (Supplementary Figure 2).