Laboratory based Retrospective Study to determine the start of SARS-CoV-2 in Patients with Severe Acute Respiratory Illness in Egypt at El-Demerdash tertiary hospitals

Title: Abstract: Purpose: COVID-19 is the most recent pandemic causing morbidity and mortality. Although a part of the pathogens causing SARI, it is unique in causing pulmonary thrombosis and lung fibrosis. Thus different management is needed. We aimed o explore the start of SARS-CoV-2 in preserved SARI samples to know the exact time of its emergence in our hospital, to conduct whole-genome sequencing in positive SARS-CoV-2 samples to define its strain. To assess the clinical characteristics of the severe respiratory infection admitted to El-Demerdash hospitals in that same period from our own file reports. Methods: We conducted a retrospective cohort study among SARI patients who were admitted to Ain Shams university hospitals. preserved nasopharyngeal& oropharyngeal swabs from 333 SARI patients were used in SARS-CoV-2 detection by RT-Real time PCR. Moreover, whole-genome sequencing of SARS-CoV-2 positive samples was performed. Clinical characteristics of the SARI patients were analyzed in the same period to show the relation to morbidity and mortality.


Introduction:
SARI is one of the clinical manifestations of COVID-19 disease, and the most lethal. Started in Brazil in the 9 th epidemiological week of the outbreak(1). But the exact start of the disease in tertiary hospitals in Egypt is still undetermined as measured by the number of patients needing hospitalization or intensive care admission due this deadly respiratory illness. While SARS-CoV-2 is under the umbrella of SARI, its disease coarse is much different with high risk for thrombosis, lung fibrosis and embolism(2, 3) As we know different age groups suffer from different pathogenic infections, children and neonates are mainly infected by Streptococcus pneumoniae and RSV in a large cohort done recently (4)while in adults influenza virus is one of the commonest causes of SARI (5). In Japan the prevalence of Streptococcal pneumoniae is 22% among the healthy children, risk factors include older siblings, older age of the child and day-care attendance (6). The increase prevalence means endemicity of the bacteria and tendency toward bacterial resistance On the other hand; different microbiological organisms cause different effect on morbidity and mortality. Also concurrent infections could exacerbate the manifestations of the disease (7).
Here in our study we examined the patients for clinical symptoms and multi-viral and multibacterial survey; who had severe respiratory illness in the Pediatrics, Internal Medicine, Geriatrics and Chest Wards and departments'-related ICUs from November 2019 to April 2020.

Materials and methods
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 6 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    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:

>hCoV-19/Egypt/MASRI-030/2020|EPI_ISL_482775|2020-04-14
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.

Ethical standards
Prior to study initiation, the study protocol was reviewed and approved by the Ethical

Results
This cohort study was conducted in Ain Shams University Hospitals on 333 patients, 273 pediatrics and 60 adults. We explored the presence of SARS-COV-2 in all these samples ,also we tested the clinical data and the effect of bacterial versus viral or coinfection on the clinical outcome.
The first COVID SARS-CoV-2 case detected was B.1.1.7, clade GR. This sample was shown with bigger font in the GH clade, which seems to be a more infectious clade than the others.  There was a statistical significant difference between the two groups regarding the mono versus co-infection as shown in figure 3.
Demographic data was analyzed for prevalence and percentage. The mean age in children was divided in two categories below or equal 2 years and above two years, as most of our pediatric sample was in those age groups. Also, as for other relevant clinical data as influenza vaccination and antiviral intake are presented in table 1.
We assessed the relation between viral infection, bacterial infection and co-infection and the various clinical comorbidities and clinical presentation and outcome in table 2.
The distribution of the study population according to their governorate place of inhabitance is described in figure 1, as this is a tertiary hospital we find patients from all across the country. But as we notice that due to the geographical area most patients are from Cairo. 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65 Regarding the clinical data and comorbidities we found that the pediatrics group had more prevalence of associated Asthma and chronic hematological disease. While adults has more prevalence of diabetes, chronic respiratory disease, chronic renal illness, immunodeficiency, chronic cardiac disease and chronic liver disease figure 2.
ICU admission and ICU transferal from ward were markedly higher in the adult group than the pediatric group as shown in table 1. Most common detected organism in swab and throat cultures was Klebsiella pneumoniae as shown in table 1.
Although the temperature >38 at presentation was not correlated with the number of total infections, the unique distribution was apparent in figure 4.
A multivariate model by logistic regression to predict the primary outcome death or discharge from the hospital was presented in table 3.

Discussion:
Our study is a retrospective cohort study assessing the cause for SARI from November 2019 till April 2020. We found that the first detected SARS-CoV-2 sample was on 14 April 2020 of a female pediatric patient 6 months of age with hematological malignancy. She presented with high-grade fever >40°C and severe cough. She didn't need ICU admission or ventilation and was isolated in pediatric ward and received medical treatment, and discharged later after improvement and negative swab.
In accordance with our study about the start of dispersion of infection of SARS-CoV-2, another Scottish retrospective study examining upper respiratory samples stored from December to  among the general population (19).
We found in our study that the comorbidities were different in the adults and the pediatric group; were chronic cardiac illness and COPD along with diabetes and renal disease were more 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65 prevalent in the adult group. On the other hand chronic hematological illness and Asthma were more prevalent in the pediatric group. Comparative to a previous SARI studies were they found that chronic cardiac disease and respiratory illness were the most common association with SARI but they didn't specify the age groups (20). Also a study conducted in Chilie found that adults are more prone to SARI when they suffered from cardiac and respiratory illnesses (5). An Egyptian study survey was conducted recently in Assuit and included pediatric and adult groups from 2010-2014 by Hatem et al.,they found that 53% of patients had co-morbidities mostly in adults not children, and in the form of chronic lung diseases 43% (21).
This indicates that co-morbidities in adults is more common than children owning to the risk factors as aging, smoking, long term illnesses, while in children most of the comorbidities is related to hereditary, nutritional or environmental risk factors (22,23).
While vaccination is slightly more prevalent in the adult group (3.1%) than the pediatric group (1.4%), there is no real difference, this could be due to low influenza vaccination rates in the high risk and vulnerable groups in Egypt; although there is ongoing plan to extend the care to overcome the limitations encountered (24).
In our study the prevalence of Streptococcal pneumoniae in nasopharyngeal and throat swabs were 43.2% in pediatrics and 10% in adults, thus more prevalent in the pediatric group. Streptococcal pneumoniae a known pathogen to cause meningitis, along with pharyngitis, appears to be common in children. Although vaccination and proper antibiotic regimens appear to be effective, the emergence of resistant strains are affecting the overall course of the infection (25). Increasing the number of siblings in the household is an important risk factor, also increase in the density of bacterial load in the nasopharyns, where both seems to be affected by the hygiene of the patient and the socioeconomic class (26). On the other hand; a recent interesting study noticed the effect of respiratory syncytial virus on increasing the rate of bacterial streptococcal co-infection in children (27), this may be attributed to the enhanced attachment to the epithelium and facilitation of immune invasion in viral infection (28).
Unfortunately; the bacterial infection in both pediatric and adults groups with Klebsiella pneumoniae was common with 129 (47.3%) cases and 22 (36.7%) cases respectively. In addition to being a cause for severe respiratory illness mandating hospitalization, this strain is more prone to multidrug resistance and complications especially in ICU patients (29).
The prediction model we used explains the effect of diabetes, which is a cause for relative immune-deficiency in adults and its association with increased infection rate in general as a predictor for increased mortality in SARI patients (30). And since most of the diabetic patients in our study are present in the adult group that explains the higher mortality in adult group (18.1%) relative to the pediatric group (7.1%) with odds ratio OR=5.7.
In addition the need for ventilation as an indication for severity of the respiratory illness was a strong predictor for mortality with OR= 316. Number of bacterial infection increased the odds of mortality as we explained the role of co-infection in affecting the immunity response to infection.

Variables
Significance OR