key: cord-0982068-3xe64vmp authors: Scarpati, Giuliana; Piazza, Ornella; Pagliano, Pasquale; Rizzo, Francesca title: COVID-19: a confirmed case of reinfection in a nurse date: 2021-07-13 journal: BMJ Case Rep DOI: 10.1136/bcr-2021-244507 sha: 871359053c807a8a6cc863be56f104c776b81054 doc_id: 982068 cord_uid: 3xe64vmp We describe the case of a 63-year-old man who is reported to have the first confirmed case of COVID-19 reinfection in Campania Region, Italy. We found that the two episodes were caused by virus strains with clearly different genome sequences. The patient, a retired nurse, had a very low level of antibodies IgG directed against the spike protein 14 days after his first Pfizer/BioNTek vaccine shot. We describe the case of a 63-year-old man who is reported to have the first confirmed case of COVID-19 reinfection in Campania Region, Italy. We found that the two episodes were caused by virus strains with clearly different genome sequences. The patient, a retired nurse, had a very low level of antibodies IgG directed against the spike protein 14 days after his first Pfizer/BioNTek vaccine shot. Worldwide, several confirmed cases of COVID-19 reinfection have been documented. 1 Perez et al 2 estimated an incidence of COVID-19 reinfections of 1 per 1000 individuals in Israel, considering that, out of 149.735 individuals with a documented positive PCR test between March 2020 and January 2021, 154 had two positive PCR tests at least 100 days apart. Reporting of reinfection cases can be difficult and their number underestimated since it is necessary to differentiate between a reinfection from a new coronavirus entering the body and a reactivation. This issue of viral reactivation or reinfection with a different strain can be resolved by sequencing of viral genome, but it is possible only if a sample, after the first episode has been obtained, kept and sequenced, and confronted with a second sample from the same patient, which had tested positive for COVID-19. The genomes of the viruses from the two samples need to be different for it to occur as a reinfection. 3 Currently, case definition of COVID-19 reinfection is lacking, but ECDC guidelines, 3 suggest considering a suspected COVID-19 reinfection case when a positive PCR or rapid antigen test (RAT) sample follow a previous positive PCR or a previous positive RAT or a previous positive serology (anti-spike IgG Ab), after more than 60 days. Kapoor et al, 4 in patients with cancer, hypothesise that the oscillating positive/negative PCR reports could be a reactivation of a dormant virus, which is commonly seen in immunosuppressed subjetcs with viruses like cytomegalovirus, herpes and Ebstein-Barr virus. To date, most of the documented SARS-CoV-2 reinfections were milder than first encounters with the virus, although some have been more harmful and people have died as a result (table 1) . Unfortunately, in patients with malignancies too, the second viral attack (reinfection or reactivation) may be more severe than the previous one, in an unpredictable way. COVID-19 treatment is not changed if a reinfection or a reactivation is known to take place and then distinguishing between reinfection or reactivation is not clinically relevant for the single patient, but the knowledge that reinfection and reactivation both exist, can help in choosing the right public health policy. In fact, even if neutralising antibodies are generated in response to SARS-CoV-2, they do not confer lifelong immunity and this limits the efficacy of strategies based on the so-called 'herd immunity'. Here, we describe the case of a 63-year-old man who is reported to have the first confirmed case of COVID-19 reinfection in Campania Region, Italy. We found that the two episodes were caused by virus strains with clearly different genome sequences. A 63-year-old male patient first acquired COVID-19 infection in March 2020, working as a nurse in a surgical ward. At that time, he had no symptoms and proved positive for COVID-19 during an epidemiological testing (14 March 2020). He was not hospitalised but isolated for prevention of onward transmission, until he tested negative twice. He was quite well until 8 months later, even if his past medical history reported chronic obstructive pulmonary disease (COPD), type II diabetes, atrial fibrillation. He got his first shot of Pfizer vaccination (figure 1) on the 13 January 2021, as offered to all the nurses, included those who retired, like our patient did in the meanwhile. On the 26 January 2021, he was admitted in hospital for respiratory failure (PaO 2 59 mm Hg, PaCO 2 29 mm Hg, pH 7.44, lactate 1,7 mm/L, respiratory rate (RR) 35). He was afebrile, with a temperature of 36°C. Nasopharyngeal swab on the 26 January 2021 demonstrated the presence of SARS-Cov-2 RNA. For the rapid worsening of his clinical presentation, the patient was admitted to our Intensive Care Unit (ICU), on his second hospital day. His pulse rate was 101 beats per minute, his blood pressure was 140/70 mmHg and his SatO 2 was 96% while he was breathing O 2 90%, by noninvasive mechanical ventilation (facial mask, PEEP 10 cmH 2 0; PS 10 cmH 2 O). The two specimens positive for SARS-CoV-2 were collected from the Salerno University Hospital Virology Lab and then analysed by whole viral genome sequencing using an amplicon panel. Illumina sequencing yielded 1 048 775 reads for the specimen collected in March 2020 (Sample A), and 3 239 835 reads for the specimen collected in January 2020 (sample B). Sequence data analysis revealed that the virus present in Sample A was a member of clade 20A (Clade GISAID O). Genomic sequence analysis identified Case report 13 At his ICU arrival, CT scan was positive (figure 3), with bilateral consolidation areas, multiple ground glass opacities, interlobular septa and intralobular lines thickening in both lungs; echocardiography showed left ventricular hypertrophy, with Ejection Fraction (EF) 60%, E/A ratio 08, Tricuspid Annular Plane Systolic Excursion (TAPSE) 10 mm, suggestive for cor pulmonale. Lymphocyte count was normal at admission and stayed within acceptable limits for the whole length of our patient's stay in ICU. C reactive protein level was elevated (17.24 mg/dL) at hospital admission (26 January 2021); total serum IgM were 116 mg/dL on the ICU admission (27 January), while IgG anti spike (COV-2 IgG) were negative (39.2 AU/mL). SARS-CoV-2 RT-PCR was performed using Allplex assay, following producer instructions and as described by Farfour et al. 6 Viral whole genome sequencing and bioinformatics analysis: library preparation was performed using the CleanPlex SARS-CoV-2 FLEX Panel (Paragon Genomics, Hayward, California, USA) for target enrichment according to the manufacturer's instructions. Briefly, multiplex PCR reactions were performed using 343 pairs of primers separated into two pools covering the entire genome of SARS-CoV-2. Illumina indexes were introduced by PCR. Library quality and concentration was assessed with 4200 TapeStation system using Agilent High Sensitivity (HS) DNA Kit (Agilent Technologies, Santa Clara, California, USA) and Qubit Fluorometer with the doublestranded DNA (dsDNA) HS Assay Kit (Life Technologies, Carlsbad, California, USA). Libraries were pooled in equimolar ratios to reach the recommended final concentration of 4 nM and sequenced on an Illumina MiSeq with 2 × 150 bp using V2 flowcell. The FASTQ files were checked for quality using FASTQC tool (https://www. bioinformatics. babraham. ac. uk/ projects/ fastqc/), and adapter sequences were removed with cutadapt V.3.2 7 using default parameters. The obtained reads were aligned on SARS-CoV-2 genome (primary assembly MN908947.3) with BWA. 7 Mutations were called using Freebayes V.1.0.2, 8 requiring a minimum coverage of 10X and low-confidence variants were removed with snippy-vcf_ filter, 9 setting the following parameters: minqual 100-mincov 10-minfrac 0.1. Consensus sequence were generated using bcftools. 10 Annotation of variants were performed using COVID-19 genome annotator, 11 while clade assignment was performed using COVIDEX 12 and Nextclade. [13] [14] [15] The two sequence are available in GISAID EpiCoV Database with the following GISAID Accession EPI_ISL_1361596 and EPI_ISL_1361597. Quantitative determination of IgG antibodies against the spike receptor-binding domain of SARS-CoV-2 in serum was performed by the SARS-CoV-2 IgG II Quant assay (ABBOTT), a chemiluminescent microparticle immunoassay, according to producer instructions. The patient's relatives have provided written informed consent for publication. Interleukin-6 (IL 6) inhibitor (tocilizumab 8 mg/kg up to a maximum of 800 mg, followed by a second dose after 12 hours) and dexamethasone 6 mg/die intravenous were administered at ICU admission, but without success. He was ventilated in prone position in spontaneous ventilation (facial mask, NIV) but after 4 days (31 January 2021) he was intubated and sedated, for the clinical worsening and his perceived suffering. He received standard therapy with neuromuscular blockade, prone positioning, and a trial of inhaled nitric oxide. Venovenous extracorporeal membrane oxygenation (ECMO) indications for this patient were collegially discussed, but in absence of local ECMO resources and of a regional ECMO referral networks to expedite patient Figure 1 Timeline of symptom onset, molecular diagnosis and ICU admission. referral and mobile ECMO retrieval, it was desolately dropped. The patient died after 11 days in ICU. Reporting the unfortunate case of this patient, the Authors signal that we, as citizens exposed to the risk of infection and physicians in care of COVID-19 19 patients, deeply need the work of dedicated genomic labs that provide useful systems for genomic situation reporting globally, in order to understand regional outbreaks and variants, that later may become dominant because of some selective advantage. SARS-CoV-2 variants may hypothetically render vaccines less effective (vaccine escape), or being associated with differences in symptoms and disease course. May the difference between the two variants reported in our patient (clade 20A (Clade GISAID O) and clade 20E (EU1) (Clade GISAID GV, the SARS-CoV-2 variant with the A222V substitution in the spike glycoprotein) mediate an altered immune response, which could explain such different clinical scenarios in the same subject? Unfortunately, we have no direct answer to this question, but in case of 20E (EU1), the variant appeared to have similar transmissibility and caused similar clinical presentations in Europe. 5 ► Consider a suspected COVID-19 reinfection case when a positive PCR follows a previous positive PCR after more than 60 days. ► Even if most of the documented SARS-CoV-2 reinfections were milder than first encounters with the virus, they can be letal. ► It is not known what level of antispike IgG is required for the efficacy of Pfizer/BioNTech mRNA vaccine. Chest CT scan at ICU admission, the patient was on spontaneous non-invasive ventilation (facial mask). What reinfections mean for COVID-19 1 to 1000 SARS-CoV-2 reinfection proportion in members of a large healthcare provider in Israel: a preliminary report European Centre for Disease Prevention and Control. Reinfection with SARS-CoV: considerations for public health response Reinfection or reactivation of Coronavirus-19 in patients with hematologic malignancies: case report series Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020 The Allplex 2019-nCoV (Seegene) assay: which performances are for SARS-CoV-2 infection diagnosis? Cutadapt removes adapter sequences from high-throughput sequencing reads Fast and accurate short read alignment with Burrows-Wheeler transform Haplotype-based variant detection from short-read sequencing Snippy: fast bacterial variant calling from NGS reads A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data Coronapp: a web application to annotate and monitor SARS-CoV-2 mutations Covidex: an ultrafast and accurate tool for virus subtyping Patient consent for publication Next of kin consent obtained.Provenance and peer review Not commissioned; externally peer reviewed.This article is made freely available for use in accordance with BMJ's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by BMJ. You may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. Giuliana Scarpati http:// orcid. org/ 0000-0002-1349-3583 Ornella Piazza http:// orcid. org/ 0000-0002-0316-5930 Francesca Rizzo http:// orcid. org/ 0000-0003-1783-5015Copyright 2021 BMJ Publishing Group. All rights reserved. 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