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Proceedings S.Z.M.C. Vol: 34(2): pp. 1-3, 2020. PSZMC-742-34-2-2020
An Approach to Screen COVID-19 Patients Using
Diagnostic Kits: With Special Reference to Pakistan
1Faiza Gul Durrani, 2Farhat Zaheer
1Consultant, Science shines Ltd., London
2Retired Chief Scientific officer (CSO), Institute of Nuclear Medicine and Oncology, Lahore
ABSTRACT
The World Health Organisation declared Covid-19 as a pandemic on 11th March 2020. The main approach to
tackle Covid-19 worldwide is to screen and provide supportive care to patients. For screening purpose RT-
qPCR- based test are used as an initial detections assay. The test is expensive, time consuming and not
suitable for mass scale screening/ confirmation requirement. A recent advancement is development of
Immunoassay procedures (liquid Phase tests or bed side 10-20 minute strip test). In order to help and
accelerate bringing life to normal after lock down, Pakistan is in dire need to develop and adopt the
immunoassay procedures for mass scale screening and confirmation of COVID-19 infection. It is cheap and
easy to perform without a lab requirement.
INTRODUCTION
An outbreak of novel Corona virus infection
(COVID - 19) in Wuhan (China), has rapidly spread
throughout the world. A current estimate indicates
total number of incidences of infected persons in the
world is 1,452,378, total deaths 83,614 and
recovered persons are 282,147 (8th April, 2020).1 In
Pakistan, the first patient was reported on 26th
February 2020 and number of incidences reported
on 8th April 2020 was 4,196 cases, deaths 60 (1.42%)
and the number of recovered cases, 467(11.1%)
According to a Ministry of Health report, the
projected number of incidences is 50,000 by the end
of April.2
Background:
Since 1960s, Corona viruses have been recognized
as one of the infection sources responsible for
common cold in humans. Six species of human
corona viruses have been identified to
date(HKU1 ,229E , NL63 ,MERS and SARS-
CoV).3 The pandemic virus has been designated as a
sister to SARS-CoVs, hence the name SARS-CoV-2
by a corona virus study group of the International
Committee on Taxonomy of viruses. The corona
viruses are enveloped viruses with a positive-sense,
single stranded RNA. The first and complete
genome sequence of COVID-19 (GenBank:
MN908947.3) and the structure of the virus’s main
protease to the Protein Data Bank (DOI:
10.2210/pdb6lu7/pdb) have been reported4. Its
genome constitutes 29891 nucleotides, encoding for
9860 amino acids. The virus has 70 percent
similarity in genetic sequence with SARS. The x-ray
crystallographic protein data bank structure
explained the glycosylated crown shape covering
(hence give the name “corona”, Latin for crown).
These spike proteins facilitate the virus to bind to
the receptors on the cells. The COVID-19 has 2 S
domains - S1 and S2. The computational analysis of
S1 and S2 has shown the importance of the S1
domain which has many unique residues to not only
bind to CD26 receptor cells but also ACE2 cells5. It
has been reported that this particular virus can infect
human respiratory epithelial cells 100-1000 times
more as compared to previous corona strains
because of its unique binding sites for ACE2
receptors. In severe cases, it can cause organ failure
and even death6.
For any viral infection/medical illness there is a set
route of diagnosis and treatment. The strategy
advised by WHO is based on screening and
providing supportive system to the patients in need.
Throughout the world, screening of the infected
individuals is essentially based upon: thermal
monitoring of the body temperature, breathing
problem, and dry cough. It is noteworthy to
mention here that clinical symptoms after infection
would appear after a period of 3-5 days of infection.
These may be delayed for two weeks, besides the
fact that some of the infected subjects may remain
asymptomatic7. Infected individuals identified by
screening are isolated at least for 14 days
(quarantine) as per WHO guidelines.
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An Approach to Screen COVID-19 Patients Using Diagnostic Kits: With Special Reference to Pakistan
Diagnostic tests for COVID-19 with special
reference to Pakistan
In Pakistan, at present in order to confirm the
COVID-19 infection, only molecular based RT-
qPCR is allowed. RT-PCR technique is very well
established in all the research institutes of the
country.17 In particular case of COVID-19, It is
performed with the kits available to the Govt.
(imported purchase or donation). Most of the kits
contain 3 assays in which the targets are Orf1 gene
(human RNA polymerase protein) E-gene (envelope
protein) and the N-gene (the nucleocapsid protein)8
so if the virus does mutate the chance of all three
targets mutation is low, however if the results of 1
or 2 of these essays is not positive then the result
needs to be revised. A possibility of a false negative
and a false positive cannot be excluded and in order
to confirm the results it needs to be repeated for
verification. Bearing in mind that the procedure is
lengthy, requires BSL-2 Lab facility and it is an
expensive test especially in view of repeat required9.
In patients with confirmed COVID-19 diagnosis, the
laboratory evaluation should be repeated to evaluate
for viral clearance prior to being released from
observation.
The possibility of a local RT-q PCR kit production
is being explored or it is in the process of validation
in Pakistan. However, the primers are not
synthesized in Pakistan. Though, at present the
requirement of tests is being managed by the
country adequately as far as possible however it is
still limited to a single test performance, with hardly
required repeat tests as mentioned above. Also, in
view of the rising number of infected patients being
identified, the country will reach an exhaustive stage
on how to manage the required number of tests.
It will not be possible to manage the required lab
workload; the country requires to be prepared
for that situation.
The diagnostic test in particular for COVID-19,
approved by WHO in its briefing (early March,
2020) was by RT-qPCR. However, WHO also
emphasized the importance of immunoassay
serological tests, which can be used as bedside tests
and will be a big breakthrough for testing service at
mass level. These immunoassay-based tests
reagents/kits have been manufactured by several
countries including USA, China, South Korea,
Ireland etc.12,13 WHO is working on its validation.
As soon as the validation is completed, these tests
will be recommended because of their specificity for
detection of antibody IgM, IgG in patients who are
in recovering phase or have had been previously
infected, 10. Moreover, the immunoassay test can
also be applied for the detection of antigen (virus),
during the first week of infection.
The detection of antibodies, (IgM and IgG) in blood
by immunochromatography procedure is simple and
straightforward. These two substances are produced
in blood after infection, usually by day 7 and 14
respectively. It requires a nitrocellulose strip with a
sample pad, and coated anti IgM and IgG pads. To
the sample pad a drop of sample (blood) is added by
a finger-prick followed by buffer. The sample and
buffer move laterally towards first IgM- antibody
pad band where IgM, if present in the sample will
bind to antibody and a colour band is produced.
Similarly, the next pad with anti-IgG coated will
identify the presence of IgG produced in blood as a
result of infection. Both IgM and IgG identify the
status of infection - present or past respectively. A
quality control colour band will also be produced at
its respective position on the strip to confirm that
the test procedure is working accurately.11
The immunoassay technique reagents are available
both for Enzyme Linked Immunosorbent Assay
(ELISA) procedure in liquid phase and Lateral Flow
Immunoassay (LFIA) for immunochromatographic
method. Bulk reagents, viz. antibody, active site
fragments of viral protein antigen, Enzyme linked
tracer for ELISA are available besides the
availability of nanoparticle linked, fluorescent
probes etc. The immunoassay kits for the detection
of viral antigen and antibody are available
internationally.12,13 As described above several
countries, including China, South Korea, UK,
Europe, and the USA are in process of producing
immunoassay kits for COVID-19 antigen/antibody
detection. The main consideration in hand in
Western countries is the validation of the assay
which is essential for quality control. It is
anticipated that assays validation will be achieved in
a few weeks’ time if not days. Once it is achieved,
there is no reason why Immunoassay technique will
not become a front-line technique and priority of
choice to be applied for mass scale screening and
for confirmation.
A comparison of the two techniques, RT-qPCR and
the immunoassay techniques are clearly indicative
that immunoassay procedure is less expensive, easy
to perform and results are available within 10-20
minutes14.
CONCLUSION
It is concluded, that Pakistan should adapt and
validate the immunoassay procedures. This would
be first step forward towards normalization after
lockdown and quarantine situation. It will be helpful
3
An Approach to Screen COVID-19 Patients Using Diagnostic Kits: With Special Reference to Pakistan
to bring the healthy recovered persons back to work,
hence normalize routines14 Furthermore, Pakistan
has the capability and expertise to produce
immunoassay-based kits as previously developed by
INMOL Lahore (PAEC) for hormones and Tumour
marker assays.15,16 Pakistan should invest to prepare
the antibody-based immunoassay kit because of its
high specificity, low in cost and easy to use
methodology.
REFERENCES
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coronavirus.jhu.edu, viewed on 8th April 2020,
2. Ashfaq, A “Corona virus: More than 50,000
COVID-19 patients expected in Pakistan by end
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sciencemediacentre.org, viewed on 1-8 April
2020
15. Zaheer, F. 1992 “Behaviour of 1-125 Labelled
Monoclonal anti TSH, and Cellulose Linked
(Solid Phase) antibody in a supersensitive TSH
IRMA “IAEA-SM-324/69: 73-81. proc. on Dev.
RIA and related procedures Vienna Austria.
16. Zaheer, F. 1992 “New perspectives for
Radioimmunoassay in developing countries
“IAEA-SM-324/71: 579-583. proc. on Dev.
RIA and related procedures Vienna Austria.
17. Durrani FG, Gul R, Sadaf S, Akhtar MW.2015
“Expression and rapid purification of
recombinant biologically active ovine growth
hormone with DsbA targeting to Escherichia
coli inner membrane”. Applied Microbiology
and Biotechnology. 99(16):6791-6801. DOI:
10.1007/s00253-015-6751-6.
The Authors:
Dr. Faiza Gul Durrani
Consultant,
Science Shines Ltd., London.
Dr. Farhat Zaheer
Retired Chief Scientific officer (CSO),
Institute of Nuclear Medicine and Oncology Lahore
(INMOL- PAEC).
Corresponding Author:
Dr. Faiza Gul Durrani
Consultant,
Science Shines Ltd., London.
E-mail: faizaguldurrani@gmail.com