A- Methodology adopted in the retrospective study:

Sample Size Determination:

500 case presented from November 2019- February 2020 will be included in explore study from
Ain shams university Hospitals

Collection of Samples:

Oropharyngeal and nasopharyngeal swabs (NP) for hospitalized SARI patients:

Clinicians in the internal medicine, chest, and pediatric hospitals will be responsible for
obtaining oropharyngeal and nasopharyngeal swabs for laboratory testing from all hospitalized
patients who meet the WHO standard SARI case definition.

Oropharyngeal swabbing:

A dry sterile tip flocked with nylon fiber 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 flexible, sterile tip flocked with nylon fiber 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 It is then slowly withdrawn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.

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 should be split into 2 pre-labeled, sterile
cryovials with the correct patient ID. One cryovial should be 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.

Samples will be tested by real time multiplex PCR using FTD- respiratory 33 kit for the
presence of a wide panel of respiratory pathogens that include: influenza A and subtypes
influenza A Pdm(H1N1) and seasonal H3, influenza B, influenza C, parainfluenza viruses 1, 2,
3 and 4; coronaviruses NL63, 229E, OC43 and HKU1; human metapneumoviruses A and B;
rhinovirus; respiratory syncytial viruses A and B ; adenovirus; enterovirus; parechovirus;
Boca virus; Pneumocystis jirovecii; Mycoplasma pneumoniae; Chlamydia pneumoniae;
Streptococcus pneumoniae; Haemophilus influenza type B (HIB assay also detects Haemophilus
parahaemolyticus which is associated with pharyngitis); Staphylococcus aureus; Moraxella
catarrhalis; Bordetella spp. (except Bordetella parapertussis), Klebsiella pneumoniae;
Legionella species and Salmonella species. Cases suspected with MERS-CoV or HPAI H5N1
according to their history of contact with birds or camels or with reference to the WHO
suspected cases of MERS-CoV or avian influenza will be tested using the CDC protocol.

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.

Sample Transfer:

All samples will be kept in Faculty of Medicine Ain Shams Research Institute- Molecular Unit
laboratories and will not be transferred to other laboratories.

B- Explore study testing for COVID-19 using ready to use RT-real time test assays - VIRAL RNA
ISOLATION: Using commercially available kits supplied by thermofisher or other recommended
companies. Work will be done in Biosafety level 2 cabinets under complete sterile conditions.
-

COVID-19 DETECTION:

Using real-time 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, thermofisher and others.

C- Genome sequencing (NGS) for possible positive covid-19 samples Ion Torrent targeted
next-generation sequencing (NGS) enables a streamlined research workflow for complete genome
sequencing and epidemiological studies of SARS-CoV-2 (the coronavirus responsible for
COVID-19). Through the use of a set of highly specific, universal coronavirus primers in
combination with a high-fidelity master mix, all genomic segments are amplified and the DNA
amplicons are sequenced on any Ion Torrent system to deliver highly accurate coronavirus
typing in under a day. The Ion ArnpliSeq SARS-CoV-2 Research Panel consists of 2 pools with
arnplicons ranging from 125275 bp iri length for complete viral genome sequencing