Covid19 Detection Methods

CONTENT
1. COVID-19 Background
2. COVID-19 Aetiology
3. Symptoms Associated with COVID-19
Infection
4. Transmission of COVID-19
5. Detection Methods
6. Comparision of different detection
methods
7. How to Protect Ourselves from
COVID-19
8. References Md. Abu Musa Al Asari
Dept. of Microbiology,
Jahangirnagar University
Email: abumusaalasari@gmail.com

COVID-19 Background
 Corona virus Disease 2019 (COVID-19) is a respiratory disease caused by
a novel strain of corona virus, which was given the interim name 2019-
nCoV by the World Health Organization (WHO), later renamed SARS-CoV-
2 by the International Committee on Taxonomy of viruses.
Coronaviruses are
-Enveloped positive-sense, RNA virus.
-Belong to the Orthocoronavirinae family.
-Cause respiratory, enteric, hepatic, and neurologic diseases.
 Although there are six known species of this virus that are known to
infect humans, however, only four are prevalent and usually cause cold-
like symptoms .These include 229E, OC43, NL63 and HKU1.
 The other two species of viruses, severe acute respiratory syndrome
coronavirus (SARS-CoV) and Middle East respiratory syndrome
coronavirus (MERS-CoV) are zoonotic viruses and have previously caused
major pandemic incidents in 2002-2003 and 2012.

Schematic labelled diagram of Coronavirus

COVID-19 Aetiology
 In December 2019, a novel severe acute respiratory syndrome coronavirus
(SARS-CoV-2) was first isolated in Wuhan, China.
 Genetic analysis conveyed that SARS-CoV-2 is closely related to SARS-CoV,
the virus that caused a pandemic in 2002-2003 and is a part of the genus
Betacoronavirus.
 It is an enveloped positive-sense RNA virus, and contains four major
structural proteins which include the spike (S), membrane (M), envelope
(E) and nucleocapsid (N).
 A recent study confirmed that angiotensin converting enzyme 2 (ACE 2), a
membrane exopeptidase, found mainly in the capillaries of the lungs, is
the receptor used by SARS-CoV-2 for entry into the human cells.
 The virus has been revealed to enter cells via endocytosis and membrane
fusion.

Symptoms Associated with COVID-19 Infection
The virus causes coronavirus disease (COVID-19) and the symptoms
associated with the disease include:
 Dry cough
 Shortness of breath
 Fever
 Headache
 Gastroenteritis
 Pneumonia
 Death in severe cases

Transmission
 The virus is thought to spread mainly from person-to-person.
– Between people who are in close contact with one another (within
about 6 feet).
– Through respiratory droplets produced when an infected person
coughs, sneezes or talks.
– These droplets can land in the mouths or noses of people who are
nearby or possibly be inhaled into the lungs.
– Some recent studies have suggested that COVID-19 may be spread by
people who are not showing symptoms.
 It can survive on contaminated surfaces and objects at room temperature
for up to six days.

Detection Methods
Molecular diagnostic tests Serologic tests
1. whole‐genome sequencing
2. real‐time reverse transcription
polymerase chain reaction
3. Loop-Mediated Isothermal
Amplification (LAMP)
4. CRISPR‐Cas system
1. Immunofluorescence Assays
2. Enzyme-Linked
Immunosorbent Assay (ELISA)
3. Microneutralization assay

 PCR is a very common scientific technique that has been widely used in
research and medicine for around 20-30 years to detect genetic
information.
 RT-PCR is a special version used when RNA is being detected and it is now
being used as a test to detect SARS-CoV-2, the virus causing COVID-19.
 This type of test has frequently been used as a frontline test for COVID-
19 as it directly tests for the presence of the virus RNA.
 RT-PCR tests are fairly quick, sensitive and reliable, capable of producing
results in 3-4 hours, although this usually takes longer if samples must first
be sent to specialised external laboratories (6-8 hours on average).
• Many diagnostic and research companies produce RT-PCR products, tests
and machines so the technology is widely available.
• Some RT-PCR tests are developed as an `all in one’ kit, reducing laboratory
handling and potential for contamination.
Reverse-Transcription Polymerase Chain Reaction (RT-PCR)

How it works?
 RT-PCR detects whether or not viral RNA is present in samples from a
patient.
 It does this by capturing and amplifying regions of the virus’ genetic
material, usually the Spike protein, N protein or Envelope.
 To measure the viral RNA, it is converted to DNA, copied many times using
repeated temperature cycles in a PCR machine and
then fluorescent markers are used to detect the virus.
 If the amount of fluorescence goes above a certain level, this confirms
that the virus is present.

1. Viral DNA is heated to 95°C
2. Separating its two strands.
3. The reaction is cooled to 55°C which
causes the separated strands to bind
with small complementary strands of
DNA in there as well as fluorescent
markers.
4. Heating the reaction up to 73°C
allows the new strands of DNA to
bind and extend producing 2X the
amount of DNA compared to that in
step 2.
5. The heating and cooling cycles (steps
1-3) are repeated around 30 times,
each time doubling the amount of
viral DNA that forms. This can then
detected by the machine.
PCR STEPS:

Interpretation
 An RT-PCR test is highly sensitive and fairly reliable if performed on a
sample from an infected part of the body whilst an active infection is
occurring.
 Positive test result: – A positive PCR result means that the person the
sample was taken from is currently infected by the virus.
 Negative test result: – A negative PCR result could mean that the person is
not currently infected by this virus, the virus is not present at the site the
sample was taken from, the sample taken was of poor quality, or that it is
too early, or too late in the infection to detect replicating virus..

Advantages
 RT-PCR is accepted by scientists and medical staff as a robust and well
documented technique.
 With RT-PCR being so common in research and medicine, the technology is
already in place to test for COVID-19.
 RT-PCR can detect current infections of disease, allowing medical staff to
determine who is currently infected and who is not.
Disadvantages
 RT-PCR relies on capturing and detecting the virus and so it is possible to miss
patients who have cleared virus and recovered from disease.
 The distribution of virus across the respiratory tract varies between patients, so
even if a person is infected, the virus may only be detectable in sputum or
nasopharyngeal swab but not necessarily at both locations at the same time.
 RT-PCR for COVID-19 can only tell if a person is currently infected with this
particular coronavirus. It can’t provide information on other diseases or
symptoms.

 Loop-mediated Isothermal Amplification (LAMP) is a similar process to RT-
PCR, but instead of using a series of temperature changes to produce
copies of the viral DNA, LAMP is conducted at a constant temperature of
60-65°C.
 The amount of DNA produced in LAMP is much higher than in RT-PCR and
a positive test result can be seen visually without requiring a machine to
read the results.
 LAMP is a newer technique compared to RT-PCR, but is technically simple
and easy for a trained scientist to perform, making it a potentially useful
technique for detection of COVID-19.
 As it is a newer technology, there is less evidence on its use, but diagnostic
companies are currently performing clinical trials to support it.
Loop-Mediated Isothermal Amplification (LAMP)

How it works?
 Like RT-PCR, the viral RNA in the sample is converted to DNA which allows it
to be copied.
 The amplification of the viral DNA using LAMP technology and reagents can
be detected when the reaction mixture is turned cloudy due to the
production of a chemical called ‘magnesium pyrophosphate’.
 As this cloudiness can be seen by the naked eye, it allows for easy diagnosis
of COVID-19 by scientists and clinicians.
 The accuracy of the results can be improved by using special
fluorescent dyes or colour changing dyes in the reaction mixture.
 As the dyes interact with the viral DNA, the intensity of the light or colour
change can be measured to give the approximate number of
viral RNA molecules that were initially in the sample.

Interpretation
Like RT-PCR, LAMP detects whether or not viral RNA is present in samples
from a patient. It does this by capturing and amplifying regions of the
virus’ genetic material, usually the Spike protein, N protein, Envelope or
multiple regions at once (see virus diagram in RT-PCR section above).
 Positive test result: – A positive LAMP result means that the person the
sample was taken from is currently infected by the virus.
 Negative test result: – A negative LAMP result could mean that the person
is not currently infected by this virus, the virus is not present at the site
the sample was taken from, or that it is too early, or too late in the
infection to detect replicating virus.

Advantages:
 LAMP is a quick technique that can produce results in 2-3 hours.
 Results can be read by eye.
 The simple and cheap method can be done within hospital laboratories, improving
the time between taking a sample and getting a diagnosis.
 LAMP can detect current infections of disease, allowing medical staff to determine
who is currently infected and who is not.
Disadvantages:
 The technology is newer than RT-PCR and does not have such a large background of
research behind it. Tests using LAMP technology for COVID-19 are still being assessed
in clinical settings.
 The science behind building these tests is more difficult than RT-PCR.
 LAMP tests rely on capturing and detecting the virus and so it is possible to miss
patients who have cleared virus and recovered from disease.
 LAMP tests for COVID-19 can only tell if a person is currently infected with this
particular coronavirus. It can’t provide information on other diseases or symptoms
and does not tell staff if a patient has been previously infected with the virus or if a
patient has any immunity to the virus.

Whole‐genome Sequencing
 Whole‐genome sequencing is one of the most comprehensive approaches
for the identification of the viral Nucleic Acids.
 The first complete SARS‐CoV‐2 genome sequence was uploaded on
GenBank (accession number MN908947) by Zhang et al. on January 5,
2020.
 Five typical open reading frames (ORFs) were identified in the SARS‐CoV‐2
genome: ORF1ab polyprotein (7096 amino acids), spike glycoprotein (1273
amino acids), envelope protein (75 amino acids), membrane protein (222
amino acids), and nucleocapsid protein (419 amino acids).
 Whole-genome sequencing can be done by Illumina or Nanopore
platforms.
 As whole‐genome sequencing is relatively expensive, time consuming, and
complicated, it is unsuited for urgent and large‐scale testing.

CRISPR‐Cas system
 The clustered regularly interspaced short palindromic repeats
(CRISPR)‐Cas system is used in another promising method for NA
detection.
 Cas13a (previously known as C2c2) can be effectively reprogrammed with
CRISPR RNAs to provide a platform for specific RNA sensing.
 Upon recognition of its RNA target, activated Cas13a engages in
“collateral” cleavage of the nearby nontargeted RNAs.
 Zhang and Collins' laboratories have collaborated to develop the
SHERLOCK method that is based on the CRISPR/Cas13a system.
 This method uses the reverse transcriptase recombinase polymerase
amplification coupled with T7 transcription to amplify the target RNA
fragments of Zika virus or Dengue virus.
 These target RNA sequences further activate Cas13a, and the activated
Cas13a then cleaves the reporter RNA (quenched fluorescent RNA)
allowing for real‐time detection of the target RNA.
 Therefore, this SHERLOCK method could theoretically be developed as a
rapid detection method for SARS‐CoV‐2.

Immunofluorescence Assays
 The next method used to detect viruses in a sample are immunofluorescence
assays. It has been used extensively to directly detect a variety of viral antigens.
 Immunofluorescence use antiviral antibodies to detect viral antigens in tissue
sections or infected cells.
 Infected cells such as those from the mucous membrane of the upper respiratory
tract or cells that are present in the mucus aspirated from the nasopharynx can be
used.
 Immunofluorescence assays use a fluorescent label that is conjugated to the
antiviral antibody, which is known as direct immunofluorescence or to an anti-
antibody, known as indirect immunofluorescence.
 The amount of fluorescence seen can be viewed with UV light.
 The amount of binding of the antibody to the antigen is directly correlated with
the amount of fluorescence produced.
 The antigen(s) detected are expressed only when the virus is actively replicating;
therefore, such tests are best used to identify acute or early infection.
 The sensitivity of these tests might be expected to vary from 34% to 80%.
 WHO does not currently recommend the use of antigen-detecting rapid diagnostic
tests for patient care

Enzyme-Linked Immunosorbent Assay (ELISA)
 ELISA is the final detection method that can be used to detect if a patient
has been infected with SARS-CoV-2.
 ELISA can detect antibodies or antigens in a sample. If a patient has been
infected with SARS-CoV-2, there will be a presence of viral antigens which
can be sampled and detected.
 Antibodies specific to the viral antigens are bound to a plastic surface, and
the sample is overlaid. If the sample is positive for viral antigens, they will
bind to the specific antibodies. After, a second antibody tagged with a
marker is added.
 If the sample contains the viral antigens it will result in a positive reaction
and will be detected by the marker changing colour when the appropriate
substrate is added.
 If no viral antigen is present in the sample, no colour change will occur.
 The antibodies of SARS-CoV-2 that can be used in an ELISA detection kit
include the spike antibody, envelope antibody, membrane antibody or
nucleocapsid antibody.
 If a clinical sample is determined to be antibody-positive by either ELISA,
CDC recommend to use the microneutralization test to confirm the
positive result.

Advantages:
 ELISA is a simple and cheap laboratory technique.
 ELISA is well established and documented within science and medicine.
 Results can typically be produced within 1 to 3 hours of collecting a
patient sample.
 Because it is so quick to perform it can be done in a hospital laboratory,
cutting down the time to diagnosis.
 ELISA testing can be completed on multiple samples at once, so it can be
used for rapid testing scaled up to test larger numbers of patients.
Disadvantages:
 ELISA tests are not yet well established for SARS-CoV-2 COVID-19 testing,
although many companies are working hard to produce them and test
them in patients.

Microneutralization assay
 The microneutralization assay is a highly specific confirmatory test used to measure
neutralizing antibodies, or antibodies that can neutralize virus.
 This method is considered a gold standard for detection of specific antibodies in
serum samples.
 However, compared with the ELISA, the microneutralization assay is labor-intensive
and time-consuming, requiring at least 5 days before results are available.
 Interpretation:
• If a clinical sample is positive by either ELISA, and positive by
microneutralization, the specimen is determined to be confirmed positive.
• If a clinical sample is positive by both ELISAs, and negative by
microneutralization, the sample is determined to be indeterminate.
• If a clinical sample is positive by only one ELISA, and negative by
microneutralization, the sample is determined to be negative.
• If a clinical sample is negative by both ELISAS, the sample is determined
negative.
 In the end, a final determination of a confirmed positive serology result requires a
positive ELISA test and confirmation by microneutralization assay.

Lateral Flow / Colloidal Gold Immunochromatography
 Lateral flow assays use the same technology commonly used for pregnancy tests.
 Lateral flow tests can detect antibody to virus from patient blood indicating that the
patient has COVID-19 or has recovered from COVID-19. Less commonly, lateral flow
tests can be used to detect the presence of active virus by detecting virus proteins
directly.
 These types of tests work detect the patient’s immune antibody response to the virus
or viral antigenic proteins.
 Antibody lateral flow immunoassays can be designed to detect IgM or IgG alone or
both together.
 Antigen lateral flow immunoassays detect the virus directly without the amplification
steps of RT-PCR and LAMP, and like those tests are only able to detect current active
viral infection but not past infection.
 Lateral flow antibody tests can be completed rapidly and the tests can be produced
cheaply, so multiple diagnostics companies are working hard to develop lateral flow
tests for SARS-CoV-2.
 A major advantage for this type of test is also the ability to see if patients are currently
infected or have recovered from COVID-19 even if they have fully recovered and
cleared the virus months ago. However, it cannot distinguish between an active and a
previous infection.

Advantages:
 Lateral flow assays are extremely quick per patient, giving results in just 15
minutes.
 Testing levels of antibody in blood allows a single patient sample from one
accessible part of the body where sampling is non-invasive to be tested for
presence of virus.
 These tests require very little training to perform and don’t rely on specialist
laboratories or scientists to analyse.
Disadvantages:
 The technology is new and the evidence for its accuracy in coronavirus
diagnosis is still being evaluated.
 So far, available lateral flow tests can only determine if a patient has at some
point been infected with COVID-19. Further testing would be needed to
check if a patient is currently infected. Future versions of this technology
might allow clinicians to detect current infections.
 Lateral flow tests are more expensive and time consuming for large batch
testing than specialist laboratory based antibody tests such as ELISA.

Chest X-Ray
 A chest CT is another detection method for determining the presence of
SARS-CoV-2 in a patient who’s lab results have returned as negative
despite the patient having the associated symptoms with the virus.
 Specific features that are associated with the presence of SARS-CoV-2
include ground glass opacities in the lungs which are caused by increased
lung opacity through which the vessels and bronchial structures can still
be seen.
 In addition, interstitial thickening or partial collapse of lung alveoli can also
be seen.

Comparision of Different Detection Methods

Molecular diagnostic tests vs Serologic tests
Molecular diagnostic tests Serologic tests
Objective of the test Detection of the virus presence in
the organism
Detection of the immune response
to the virus
What does it look for? viral genetic material (RNA) or viral
antigen
immune response (antibodies)
against the virus in the patients’
blood.
What does a positive
test mean?
The virus is present in the patient. The patient has been exposed to
the virus and is either recovering
or has already recovered.
What is the test used
for?
To know whether a patient is
currently infected by the SARS-
CoV-2.
To know whether a patient has
been exposed to the SARS-CoV-2
and is therefore protected against
new infections (and may not
spread the disease anymore).

ELISA tests vs Immunochromatographic assays
ELISA tests Immunochromatographic assays (rapid
tests)
Pros i. More precise than
immunochromatographic assays.
ii. Provides a quantitative
information (concentration in
antibodies).
i. Less resource intensive than ELISA
tests
ii. Could be performed at point of care
once the technique is fully validated
(could potentially be sold to the
general public).
iii. Rapid (10 to 30 minutes)
Cons i. Needs to be performed in a lab
ii. Resource Intensive (1 to 5 hours)
i. Provides only a qualitative
information (presence or not of
antibodies)

RT-PCR vs Direct antigen detection
RT-PCR
Direct antigen detection (still under
development)
Looks for presence of viral genetic
material (RNA)
presence of viral antigens
Pros
i. If done properly, the
technique is very sensitive
and specific
ii. “Fast RT-PCR” can be used
at point of care
i. Simple
ii. Rapid
iii. Could be used at point of care.
Cons
i. Labour intensive
ii. Majority of tests still need
to be processed in a lab
iii. Risk of false negative
Complex to develop.

How to Protect Ourselves from COVID-19
As there is no vaccine developed yet and no perfect drug to cure, prevention
is the only solution. To protect ourselves we should-
 Wash hand with soap and water for at least 20 seconds especially after
you have been in a public place, or after blowing your nose, coughing, or
sneezing.
 If soap and water are not readily available, use a hand sanitizer that
contains at least 60% alcohol. Cover all surfaces of hands and rub them
together until they feel dry.
 Avoid touching your eyes, nose, and mouth with unwashed hands.
• Avoid close contact with people who are sick, even inside your home.
• Put distance between yourself and other people outside of your home.
• Cover your mouth and nose with a cloth face cover when around others
• Cover coughs and sneezes.

 Clean AND disinfect frequently touched surfaces daily. This includes
tables, doorknobs, light switches, countertops, handles, desks, phones,
keyboards, toilets, faucets, and sinks.
 If surfaces are dirty, clean them. Use detergent or soap and water prior to
disinfection.
 Be alert for symptoms. Watch for fever, cough, shortness of breath,
or other symptoms of COVID-19.
 Take your temperature if symptoms develop
 Follow CDC guidance if symptoms develop.
How to Protect Ourselves from COVID-19

References
www.who.int
www.cdc.gov
www.assaygenie.com
www.cebm.net

Covid19 Detection Methods

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