Explore study: Retrospective Cohort study to show the start of SARS-CoV-2 in patients with severe respiratory illness by real-time PCR

Aim: to know the start of COVID-19 in Ain shams University hospitals (El-Demerdash) in Severe acute respiratory illness. Materials and methods: retrospective collection of data records and previously collected nasopharyngeal and oropharyngeal sample stored in our lab for determining the start of COVID-19 in our hospitals, using RT-PCR and postive samples we will conduct Sequencing analysis and photogenic analysis. Also epidemiological and data study of the negatively screened patients, to determine the most common causes of SARI and the causes of mortality in this category of patients


Introduction
The 2019-20 coronavirus pandemic is an ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [1] The outbreak was rst deaths. [4] More than 297,000 people have recovered. [5] Medical Ain Shams Research Institute (MASRI) is conducting a surveillance project internationally with other Eastern Mediterranean region countries, we have gathered about 300 samples (nasopharangeal& oropharengeal), these samples underwent molecular Lab evaluation of COVID-19, these samples are very well preserved, with fully collected epidemiological and lab data. Due to the emerging situation of the COVID-19 pandemic we nd it mandatory to retest those samples collected since November 2019 for COVID-19 to assess if this COVID-19 was present earlier than we thought and if so we can relate its presence to the epidemiological data we had obtained earlier, moreover if COVID -19 is detected we can move step forward for next generation sequencing of the positive samples to de ne the most dominant strain found in Egyptians and further compare our strain or strains to other countries.

Reagents
Using commercially available kits supplied by thermo sher for RT-realtime test assays -VIRAL RNA ISOLATION Equipment Ion Torrent targeted next-generation sequencing (NGS) Procedure 1-Previously collected data and samples: Oropharyngeal and nasopharyngeal swabs (NP) for hospitalized SARI patients: Data and samples of oropharyngeal and nasopharyngeal swabs for laboratory testing obtained previously from all hospitalized patients who meet the WHO standard SARI case de nition.
Oropharyngeal swabbing: A dry sterile tip ocked with nylon ber swab applicator should be used to swab the posterior pharynx.
The swab should be placed in a 15 ML centrifuge tube labeled with the patient unique ID and containing 2mL viral transport media (VTM). The applicator stick is then cut off.
Nasopharyngeal swabbing: A exible, sterile tip ocked with nylon ber swab applicator is inserted into the nostril and back to the nasopharynx and left in place for a few seconds. It is then slowly withdrawn with a rotating motion. For SARI patients, the swab is put into the same centrifuge tube as the oropharyngeal swab labeled with the patient's unique ID and the shaft is cut. The 15 ml tube containing the swab/s should be carefully transported to the hospital laboratory as soon as possible in cold chain.

2-Sample processing:
The received swabs inside the 15 ml tube should be agitated vigorously for 10 seconds using a vortex mixer to free cells from the swab tip, and then both swabs should be removed from the tube and discarded using a forceps. The VTM was split into 2 pre-labeled, sterile cryovials with the correct patient ID. One cryovial was immediately placed in a freezer (-70°C), while the other cryovial will be used the molecular testing by real time PCR at MASRI molecular laboratory.

3-Sample storage:
The second aliquot of the samples will be stored in freezers (-70°C) at MASRI MOLECULAR laboratory for External Quality Assurance (EQA) by on site testing through random selection of samples with different results and retest them by other NAMRU-3 laboratory expertise in MASRI molecular labs ; the samples can also be used for further testing by NGS or virus isolation in MASRI molecular laboratories.

4-Sample Transfer:
All samples will be kept in MEDICAL AIN SHAMS RESEARCH INSTITUTE laboratories and will not be transferred to other laboratories. 5-Explore study testing for COVID-19 using ready to use RT-realtime test assays -VIRAL RNA ISOLATION: Using commercially available kits supplied by thermo sher or other recommended companies. Work will be done in Biosafety level 2 cabinets under complete sterile conditions. -COVID-19 DETECTION : Using realtime reverse transcription polymerase chain reaction RT-PCR } the test can be done on respiratory samples obtained by various methods, including nasopharyngeal swab or sputum sample. Results are generally available within a few hours. Molecular methods leverage polymerase chain reaction (PCR) along with nucleic acid tests, and other advanced analytical techniques, to detect the genetic material of the virus using real-time reverse transcription polymerase chain reaction for diagnostic purposes. Commercially available kits supplied by hvd , thermo sher and others. Anticipated Results