key: cord-0924318-tcv6yqkm authors: Musso, Nicolò; Maugeri, Jessica Giuseppina; Bongiorno, Dafne; Stracquadanio, Stefano; Bartoloni, Giovanni; Stefani, Stefania; Di Stefano, Emanuela Daniela title: SARS-CoV-2’s high rate of genetic mutation under immune selective pressure: from oropharyngeal B.1.1.7 to intrapulmonary B.1.533 in a post-vaccine patient date: 2022-03-02 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.02.044 sha: a76552b313fccbd6fb8e773924bf889a1ed09458 doc_id: 924318 cord_uid: tcv6yqkm This is the case report of an 84-year-old man affected by COVID-19 between the two shots of vaccinal procedure, with negative exitus. We analysed nasopharyngeal samples of viral RNA collected during the disease and nasopharyngeal and lung samples collected post-mortem by RT-LAMP PCR and Next Generation Sequencing (NGS). NGS results were analysed with different bioinformatic tools to define virus lineages and the related SNPs. Both the lung and nasopharyngeal samples tested positive for SARS-CoV-2 on RT-LAMP. The bioinformatic analysis identified two viral RNAs from the nasal swabs as belonging to the B.1.1.7 lineage, whereas the RNA collected from the lung was shown to belong to the B.1.533 lineage. This genetic observation suggested that the SARS-CoV-2 virus tends to change under selective pressure. The high mutation rate of ORFa1b, containing a replicase gene, was a biological image of a complex viral survival system. This is the case report of an 84-year-old man with a medical history of chronic kidney disease, dyslipidaemia, systemic arterial hypertension, coronary and peripheral arterial dis-ease, admitted to our unit with dyspnoea and suspected COVID-19 disease contracted after partial vaccination ( Figure 1A ). On 11 April 2021, the patient received his first COVID-19 vaccine dose (Pfizer mRNABNT162B2). On around 1 May, he started developing an asymptomatic infection after contact with relatives. With this being unknown, he received his second vaccine dose. Five day later, a fever appeared. On 10 May, the patient worsened and was transferred to the COVID ED-Hub. Evolution of clinical condition, labs results and therapy at T 0 , T 24 , T 72 , and T 84 are listed in Figure S1 . The RT-qPCR gave a positive result, while the chest X-ray showed decreased lung transparency with submantellar thickening in the mid-lower field bilaterally ( Figure 1C ). He immediately received non-invasive ventilation (NIV) with protective settings and FiO 2 60%. An improvement in dyspnoea was observed. Seven hours after entering the hub, the patient was admitted to the COVID-19 Pneumology Department, where ventilatory support was converted to facial mask with reservoir. Twenty-four hours after admission, he showed severe respiratory failure with signs of microcirculatory distress -although with mild dyspnoeaat BGA, as well as persistent lymphocytopenia and inflammation. At 72 hours, the patient was transferred to the ICU with signs of worsening and severe dyspnoea, and received NIV (Rochwerg et al., 2017) in continuous positive airway pressure mode (8 cmH 2 O, FiO 2 80%). The clinical condition progressed to Critical COVID-19 (Di Giacinto et al., 2020) . At 84 hours, upon progression to severe hypoxemia and severe Acute Respiratory Distress Syndrome (ARDS Definition Task Force, 2012), the labs revealed persistent lympho-cytopenia and systemic inflammation still in the absence of bacterial superinfections identifying a IV Stage Critical COVID-19 as indicated by the Italian Society of Anesthesia Analgesia Resuscitation and Intensive Care (SIAARTI) guidelines 2020. Chest X-ray reported increased ground-glass opacity with a submantellar seat in the mid-lower field of the left lung with ipsilateral pleural veil ( Figure 1C ). The patient was intubated and switched to mechanical ventilation with maximally protective settings and FiO 2 100%. Respiratory mechanics measurements showed lung stiffness (Gattinoni et al., 1987) . Thirteen hours after ICU admission, the signs of multiple organ dysfunction suggested a progression to Septic Shock, already reported as possibly related to Critical Covid-19 (Critical COVID-19, VI Stage. SIAARTI 2020), with hypoxia and hypercarbia, and in less than 96 hours, the patient died from an episode of non-shockable rhythm. Autopsy was performed on multi-tissue samples (Musso et al., 2021) . Histology displayed the typical hallmarks of advanced COVID-19, specifically in the lung parenchyma, as well as major vascular co-morbidities in the coronary tree and myocardium. Pulmonary changes included diffuse emphysematous findings with alveolar spaces filled with pneumocytes, CD68+ histiocytes and syncytial TTF1+ pneumocytes ( Figure 1D , panels A and B), with evidence of diffuse alveolar damage and intra-alveolar fibrin deposition consisting in hyaline membranes. The inflammatory infiltrate was relatively modest and consisted mainly in clusters of CD8+ macrophages ( Figure 1D , panel C), lymphocytes andto a lesser extent -CD4+ cells. Multifocal occlusive thrombosis in the pulmonary arterioles ( Figure 1D , panel D) and severe occlusive coronary atherosclerosis completed the pathological picture. At hospital admission, a nasopharyngeal swab was collected and tested by RT-qPCR (SARS-CoV-2 Assays Allplex TM SeeGene INC.) to perform a four-gene molecular diagnosis of SARS-CoV-2 infection. Another nasopharyngeal swab and a lung sample were collected on autopsy to be tested by next generation sequencing (NGS) and compared with the previous nasopharyngeal sample. RNA was extracted using the standard procedure (Musso et al., 2021; Musso et al., 2020) and all samples were also tested by RT-LAMP. RT-LAMP was performed using the SARS-CoV-2 POC kit (Enbiotech, Cat. EBT 102-48) in the ICGENE Health (Enbiotech SRL, Cat. EBT 806) system according to the manufacturer's protocols . (Hadfield et al., 2018) to identify and confirm the correct lineage, and to GISAID (Elbe et al., 2017) to obtain a cluster of SNPs from these genomic sequences. Both the lung and the two nasopharyngeal samples tested positive for SARS-CoV-2 on RT-LAMP within 45 and 30 minutes of reaction, respectively. The bioinformatic analysis identified the two viral RNAs from the nasal swabs as belonging to the B.1.1.7 lineage, whereas the RNA collected from the lung was shown to belong to B.1.533. All NGS results were obtained with high coverage (over six million readings per sample, Figure 1B ). On 10 May, when the patient's oropharyngeal swab was analysed, the result of the RT-qPCR was positive but with a very high CT (>36), indicating a low viral load in the oropharyngeal cavity. However, this finding is inconsistent with the lung damage seen at the first chest X-ray. One explanation could be the "RNA fragmentation" 5 in the nasal cavity, probably due to antibody reaction. In the thirteen days of intra-pulmonary residence, the virus managed to develop "intra-host specific rearrangements" (Voloch et al., 2021; Wang et al., 2021) , i.e., mutations capable of making it more aggressive, such as T4087I in ORF1ab and S116T in the Spike gene. This evolution could explain the difference in lineage between the two different anatomical location of the same samples: B.1.1.7 for the nasopharyngeal virus and B.1.533 for the lung virus, with the latter being apparently more aggressive ( Figure 1B ). In particular, B.1.533 is currently not widespread in Italy, as it is in the rest of the world, though with low percentages. This increased aggressiveness could explain the presence of both clones in the lung sample, whereas only the B.1.1.7 clone was observed in the oropharyngeal sample (O'Toole et al., 2021) . The scenario described was the one of severe respiratory failure from diffuse alveolar damage associated with lymphomonocyte interstitial pneumonia and thrombotic diathesis in the lung arterioles, consistent with the reported etiopathogenesis of SARS-CoV-2 infection. The immune reaction was a combination of vaccine and immune response following infection with the SARS-CoV-2 virus, but the presence of antibodies did not lead to the disruption of the viral RNA before this could cause pulmonary infection; quite on the contrary, it accelerated the normal process of "intra-host specific rearrangement", as shown by the presence of a new intra-pulmonary lineage characterized by five, worldwide low expressed SNPs (<2%): T1022I-I23N-T145I-A185S-S116T. T1022I and I23N (in ORF1ab) have an effect on the oligomerization interface; the last SNP, also present in ORF1ab, affects the antibody recognition site/oligomerization interface (Table 1 ). The two lineages differ from each other as well as from the reference genome as follows: 11 amino-acid substitutions in Table S1 ). This genetic observation suggested that the SARS-CoV-2 virus tends to change under selective pressure. The high mutation rate of ORFa1b, containing a replicase gene, was a biological image of a complex viral survival system. 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Virus Evol Official ERS/ATS clinical practice guidelines: noninvasive ventilation for acute respiratory failure Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) as a user-friendly system to detect SARS-CoV-2 infection: a multicentric study Intra-host evolution during SARS-CoV-2 prolonged infection Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients The authors wish to thank Prof. Cristoforo Pomara as the pathologist in charge of the autopsy and Dr. Gregoria Bufalino, who assisted the patient in our case report. We also wish to thank PharmaTranslated (http://www.pharmatranslated.com/), and in particular Silvia Montanari for language support.