This is a retrospective cohort study from our hospital records, where we used previously collected stored samples from patients admitted to the specific hospital departments of Ain shams university hospitals (Pediatric hospital, Geriatric hospital, Internal medicine department, Chest department, Pediatrics ICU, Chest ICU and Internal medicine ICU) that are responsible for the management of SARI. The patients are included when they fulfilled the criteria for acute severe respiratory infection, and followed till the end of hospitalization. The clinical outcome is followed either; discharge, transferal or death. We used sequential weekly patches of stored samples to determine the first positive SARS-CoV-2 sample and the patient data of this positive sample.
We used previously collected samples from 333 patients with severe acute respiratory illness; in the period from November 2019 until April 2020, these samples were tested for the presence of SARS-COV-2 by Real-Time PCR followed by Whole viral Genome Sequencing of the positive SARA-COV-2 samples to define the strain .
SARI cases were defined as a respiratory infection with fever of 38˚C, cough, onset within the last 10 days, and hospitalization(8). Records of previously hospitalized patients with SARI provided demographic information, including age, sex, and residence through questionnaires.
Clinical Sample Collection And Processing
Between November 2020 and April 2020 Oropharyngeal and nasopharyngeal swabs (NP) from hospitalized SARI patients were collected and placed in a centrifuge tube labeled with the patient unique ID and containing 2mL viral transport media (VTM). Patients had completed a questionnaire that covered history of fever and/or respiratory symptoms, travelling history, any underlying lung disease, history of chronic or immune-compromised conditions, and final outcome. The records were used retrospectively to assess the patients’ clinical characteristics.
The received swabs inside the 15 ml tube were agitated vigorously for 10 seconds using a vortex mixer. VTM were split into 2 pre-labeled, sterile cryovials with the correct patient ID. One cryovial were immediately placed in a freezer (-80°C), while the other cryovial was used for molecular studies at Faculty of Medicine Ain Shams University Research Institute-
Viral RNA Extraction and SARS‐Cov‐2 detection by QRT-PCR:
Laboratory detection of SARS‐Cov‐2 infection was done in Medical Ain Shams Research Institute ( MASRI ) MOLECULAR GENOMIC LABS Viral RNA isolation was performed using MagMax viral/pathogen nucleic acid isolation kit (ThermoFisher Scientific, USA). Real time reverse transcription polymerase chain reaction (RT‐PCR) was used for simultaneous amplification of two target genes, including nucleocapsid protein (N), and open reading frame 1ab (ORF1ab). COVID119 detection was done using ProLab/CerTest Biotech ViaSure SARS-CoV-2. Real Time PCR detection Kit (VS-NCO296T, CerTest Biotec, S.L, Spain, Catalogue number VS-NCO213L) that was performed upon an Applied Biosystems™ 7500 Fast Real-Time PCR System following the cycling and fluorescence acquisition parameters detailed in manufacturer protocol. Five microliters of RNA that was isolated from clinical samples were used in each real time PCR reaction, with a final volume for 20 µL. Samples were processed with appropriate; negative, internal and positive controls. Samples were run in duplicate. Real-Time Detection System and analysis was done by Applied biosystem 7500 Real-Time PCR Software v2.0.
Samples were considered negative: Ct > 38 or not detected; positive: the amplification curve was s‐shaped, and the Ct value was ≤35; suspicious: the amplification curve was s‐shaped, and 35 < Ct ≤ 38, requiring reexamination. In addition, SARS‐CoV‐2 nucleic acid test positive interpretation includes, ORF1ab and N genes tested positive at the same time in the same specimen; and the ORF1ab or N gene was positive in two different samples of the same patient based on the recommendations of the National Institute for Viral Disease Control and Prevention (China).
Viral Genome sequencing for positive SARS-CoV-2 samples by targeted next-generation sequencing (NGS)
- RNA preparation ; After viral RNA isolation(previously describes, The RNA concentration was determined by a Qubit® 2.0 Fluorometer (Qubit® RNA Assay Kit, Life Technologies) . Reverse Transcription and cDNA synthesis was done after RNA extraction and assessment, RNA was reverse transcribed using the SuperScript™ VILO™ cDNA Synthesis Kit (Cat. No. 11754050) .
- NGS Library Preparation; Manual library preparation was done using the Ion AmpliSeq™ Library Kit Plus (Cat. Nos. 4488990 )following the user guide , primer pool 1 and 2 target amplification reactions were combined and amplicons Partially digested , barcode adapters were ligated and purified using the Ion Xpress™ Barcode Adapters 1–96 Kit (Cat. No. 447451). Then libraries were quantified using the Ion Library TaqMan™ Quantitation Kit (Cat. No. 4468802). Templates were prepared and chips loaded on the ion Chef instrument Using the Ion 530™ Kit – Chef (Cat. No. A34461), according to user guide .
- Ion S5 targeted NGS and analysis The libraries were sequenced on the Ion GeneStudio S5 Series System platform with an Ion AmpliSeq SARS-CoV-2 Research Panel (ThermoFisher Scientific, USA) that contains two pools with amplicons ranging from 125 bp to 275 bp in length and includes >99% of the SARS-CoV-2 genome, covering all serotypes. A complete genome (29903 nucleotides) was assembled, with 0.13% unique mutations to the other viral genomes. Using BLAST against NCBI betacoronavirus database, the closets matches are several sequences bit score of 33479, including for example, isolate SARS-CoV-2/human/USA/VA-DCLS-0556/2020 (99.9%), accession (MT739463). The assembled genome along with the other SAR-CoV-2 genomes obtained and clustered from GISAID were aligned using MAFFT(9).
- Phylogenetic construction: A maximum likelihood phylogenetic tree was constructed using FastTree (10, 11) under the general time reversible (GTR) model(12) with gamma distribution (i.e., GTR + ) for the sites rates.
SARS‐Cov‐2 nucleic acid tests were done for all the collected swab samples (n=333), but only one case was positive for the test.
This positive case was a female patient 6 months old, her main presenting symptom was fever, and she had hematological malignancy. She is the first Egyptian positive case in Ain Shams University Hospitals that was confirmed on 14 April 2020 and she was negative for all other examined respiratory pathogens. We submitted the genome data on the GISAID website accession number:
- Reports of results of the routine nasopharyngeal swab and throat swab that were taken from all the patients with suspected SARI for culture analysis were included for statistical analysis.
- Reports of previous detection of viral infection was through lab and clinical data, where the patients were assessed for low neutrophil count and high lymphocytic count, exaggeration of fever or cough after bacterial infection is diagnosed or associated enlarged lymph nodes.
Prior to study initiation, the study protocol was reviewed and approved by the Ethical Committee of AinShams University. Reports from hospital records were used. Samples used in this study were previously ethically approved with informed patients’ consent in an ongoing project: (We have gathered nasopharyngeal& oropharyngeal swabs from 333 SARI patients which was used for molecular detection of 33 respiratory pathogenic (viral & bacterial) targets). Specific national laws have been observed
The data was analyzed using SPSS version 23.0 from IBM (IBM Corporation, Armonk, NY, USA) and Python Programming language 3.8.3. Graphs were drawn using Microsoft Word.
For numerical values median and interquartile range were calculated. For categorical values number and percentage are presented. P value was considered significant if <0.05. Fisher’s Exact test was done for cross tabulation of binary data in between two groups, while Chi square test was done for binary data with more that two groups, and Chi Square for trend test for ordered categorical data.
Multivariate analysis was done using binary logistic regression to formulate a predictive model for the clinical outcome.