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OpenWHO: Clinical Management of patients with COVID-19: Investigations and care of mild, moderate and sever
disease

Module 1: Laboratory considerations in COVID -19

Presenter: Celine Barnadas

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[Slide #1] Welcome to the module on Laboratory Considerations for COVID-19. I am Dr. Celine Barnadas
from the Public Health Laboratory's Training Team at WHO headquarters. This module is part of the WHO
COVID-19 Clinical Management Course series.

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[Slide #2] At the end of this module, you should be able to describe when to consider testing for SARS-CoV-
2. To describe different diagnostic tests for SARS-CoV-2 and to describe laboratory abnormalities commonly
seen in patients with COVID-19.

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[Slide #3] So there are many reasons to test for SARS-CoV-2, such as individual patient management and
public health reasons. A health provider may order SARS-CoV-2 test in order to confirm an active infection in
a patient who are clinically suspected to have COVID-19. That is, in someone who meets standard case
definition of a suspected COVID-19 case. You can see the link on this side for the WHO global case definition.
Another reason a clinician may order SARS-CoV-2 test is to determine current or past exposure to the virus
for appropriate cohorting of patients in a health facility. It is important to keep suspected and confirmed patients
at an appropriate distance from patients who are not suspected to have SARS-CoV-2 infection. Some public
health reasons to test for SARS-CoV-2 include screening of contacts of person with confirm COVID-19 and
other screening persons. Different testing objectives require different testing methods. Please see the WHO
guidance linked on these slides for more details.

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[Slide #4] When a patient with symptoms listed in the standardized case definition presents to health provider,
promptly consider testing for SARS-CoV-2. Please see the link on this slide for the WHO global case definition.
Symptoms of COVID-19 overlap with many other infectious and non-infectious etiologies that the patient may
have. Build a broad differential diagnosis based on setting and patient factors. That is, take syndromic efforts
to diagnosing COVID-19. A positive test for another pathogen does not rule out a SARS-CoV-2 infection and
a positive test for SARS-CoV-2 does not rule out co-infection with another pathogen or another etiology for the
patient's symptoms.

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[Slide #5] Co-infections with other respiratory pathogens, such as viral, bacterial, fungal infections have been
founding COVID-19 patients. As mentioned before. A positive taste for another pathogen does not rule out a
SARS-CoV-2 infection and vice versa. Testing for additional pathogens should be performed according to local

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OpenWHO: Clinical Management of patients with COVID-19: Investigations and care of mild, moderate and sever
disease

guidance. For example, in malaria-endemic areas, patients with fever should be tested for malaria or other co-
infections and treated as appropriate. In endemic settings, arbovirus infections such as dengue, chikungunya,
zika, yellow fever should also be considered in the differential diagnosis of undifferentiated febrile illness,
particularly when thrombocytopenia is present. Similarly, influenza or other respiratory viruses should also be
considered in the different shoulder if a season is appropriate. For more on co-infections, view the module on
Co-infections and use of antimicrobials in COVID-19, part of this course series.

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[Slide #6] There are three major types of SARS-CoV-2 tests available, molecular test, antigen tests and
antibody tests. Molecular and antigen test detect current SARS-CoV-2 to infection. Antibody tests also called
serology test asses for past infections. Molecular test detect viral genetic material by amplifying small amount
of viral genome or RNA, until it reaches detectable levels. This test is usually done in a laboratory. Antigen
tests detect viral proteins by capturing antigens on the test strip that changes in color if present. These tests
are generally done at the point of care. And finally, antibody tests look for antibodies that patients develop in
response to infection by capturing antibodies on a test trip or in a tube that changes colors if present. These
tests can be done in a laboratory or at the point of care. However, based on current evidence, WHO
recommends the use of this new point of care on your diagnostic test only in research settings. They should
not be used in another setting, including for clinical decision-making until evidence supporting news for specific
indication is available.

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[Slide #7] The different tests described in the previous slide detect different parts of the SARS-CoV-2 virus.
Here you see a simplified schematic representation of SARS-CoV-2 RNA and proteins and human antibodies
targeting SARS-CoV-2. As pictured, molecular tests detect viral RNA. The blue string-like paths visualized on
this slide. Antigen tests detect viral proteins, visualize as the spokes on the virus on this slide.

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[Slide #8] Finally, antibody tests detect viral specific human antibodies as visualized by the blue Y like figure
on this slide. There are additional lab tests being used in the COVID-19 public health response. This include
sequencing and culture. The sequencing allows us to detect SARS-CoV-2 variants by identifying mutations or
changes in the composition of the viral genome. It is mainly used for public health surveillance and research.
Then we also have culture. Culture allows us to assess the effect of drugs or antibodies on virus growth. Both
of these tests must be done in specialized laboratories.

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[Slide #9] The probability of detecting SARS-CoV-2 with a different type of test described depends on the time
of the course of infection in which a specimen was taken. This graph shows that antigen tests are best suited
to detect infection at the time of or just after symptom onset. Molecular tests are best suited to detect infection
at about two weeks after symptom onset. Antibody test, detect infection several weeks after symptom onset.

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OpenWHO: Clinical Management of patients with COVID-19: Investigations and care of mild, moderate and sever
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This is the reason why they are not the test of choice to detect acute infections. Molecular and antigen tests
can detect virus before symptom onset. So how should a clinician choose which tests to order?

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[Slide #10] In general, molecular test or nucleic acid amplification tests such as RT-PCR is a test of choice for
detecting SARS-CoV-2 in a patient who meets clinical criteria for COVID-19. If the tests result negative, but
there is continued clinical suspicion for COVID-19, resample and repeat the test. If the tests result negative
again and clinical suspicion remains, consider the use of serology test. Once a patient in COVID-19 is
suspected, has tested positive for SARS-CoV-2, he or she may be deemed a confirmed case.

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[Slide #11] RT-PCR reverse transcription polymerase chain reaction is a type of molecular assay or nuclear
acid amplification test or so called NAAT which detects viral RNA. When performed under ideal conditions,
RT-PCR has a high sensitivity and high specificity. To clarify these terms, sensitivity refers to the percentage
of SARS-CoV-2 infected patients who are correctly identified as having SARS-CoV-2 infection. It starts with
high sensitivity, meaning there are generally a few false negative results. Specificity refers to the percentage
of non-infected people who are correctly identified as not having SARS-CoV-2 infection. Otherwise say, high
specificity means there are generally a few false positive results. A described previously, SARS-CoV-2
molecular assays can be positive regardless of presence of symptoms.

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[Slide #12] Molecular tests can be run in a lab-based setting or at the point of care. In a laboratory, RT-PCR
tests are conducted by highly trained lab personnel. RNA is extracted and purified from the patients specimen.
The RT-PCI instrument amplifies and detects RNA. The output will be determination of whether SARS-CoV-2
RNA is present as well as the Ct value. Ct value refers to the PCR cycle at which the amplification of SARS-
CoV-2 becomes detectable above background noise. The more viral RNA in the sample, the smaller the Ct.
It's very important to remember that the Ct values are not directly comparable across RT-PCR platforms. At
the point of care, molecular tests are conducted using closed systems where the sample is that added in a
cartridge where the different reaction steps will take place. Again, RNA is extracted and purified from the
patient's specimens and genes are amplified and detected. The output of the molecular test will be in the
presence or absence of SARS-CoV-2 RNA.

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[Slide #13] Rapid antigen tests are usually done at the point of care. Antigen tests detect viral proteins, not
viral genetic material. These tests should not be considered interchangeable with molecular tests and we will
see this in the next slide.

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[Slide #14] Antigen RDTs are generally less sensitive compared to NAAT assays. Remember the definition of
sensitivity. Sensitivity refers to the percentage of SARS-CoV-2 infected patients were correctly identified as
having SARS-CoV-2infection. Low sensitivity means that generally higher false negative results. Antigen RDTs

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OpenWHO: Clinical Management of patients with COVID-19: Investigations and care of mild, moderate and sever
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work best in phases with high viral load, which is generally 1 to 3 days before and within the 5-7 days after
onset of symptoms. Infections with low viral loads may be missed. Stripes represent viral loads, which would
be detected by RT-PCR assays but not using antigen RDTs.

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[Slide #15] As general recommendation for the use of antigen RDTs, follow your national or local testing
strategy which defines when and when not to use antigen RDTs. WHO advises that SARS-CoV-2 antigen RDT
testing can be considered if there is widespread community transmission of COVID-19, if the health system is
over-burdened and or if it is not possible to test all suspected cases by molecular testing methods within an
acceptable turnaround times which would be of less to 48 to 72 hours. The test used should meet minimum
performance criteria of 80% or more sensitivity and 97% or more specificity. You can refer to the WHO
guidance linked on this slide for more information.

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[Slide #16] When molecular tests or nuclear acid amplification tests such as RT-PCR are not available and
there is widespread community transmission of SARS-CoV-2, antigen rapid diagnostic test may be used. A
trained operator should take the sample. If SARS-CoV-2 is detected, the patient should be isolated, according
to their symptom severity. For example, if the patient has severe critical disease, the patient should be admitted
to the hospital. If the patient has mild or moderate disease, home or cohorted isolation in a designated center
should be considered. In the case where SARS-CoV-2 is not detected, but the concern of possible infection
remains and the negative predictive value of the test is high. Patients should be referred to ward designated
for suspected cases while other causes of symptoms are explored. If SARS-CoV-2 is not detected and the
concern of possible infection is low and the negative predictive value of the test is low, advise patients to
exercise infection control practices and consider repeat testing if symptoms progress or persist.

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[Slide #17] Now in a slightly different scenario when molecular tests or nuclear acid amplification test such as
RT-PCR are available but only in a limited capacity, and there is widespread community transmission of SARS-
CoV-2, antigen rapid diagnostic tests may also be used. A trained operator should take the sample, if SARS-
CoV-2 is detected, the patient should be isolated cohorted according to their symptom severity. For example,
if the patient has severe critical disease, the patient should be admitted to the hospital. If the patient has mild
or moderate disease, Home or cohorted isolation in a designated center should be considered. If SARS-CoV-
2 is not detected, but the concern of possible infection remains and the negative predictive value of the test is
high, patients should be referred toward designated for suspected cases while other causes of symptoms are
explored. If SARS-CoV-2 is not detected and the concern of possible infection is low and negative predictive
value of the test is low. A sample for molecular tests such as RT-PCR should be taken and the patient should
be isolated until the results are available.

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OpenWHO: Clinical Management of patients with COVID-19: Investigations and care of mild, moderate and sever
disease

[Slide #18] We're now going to talk about serology. Serology can detect viral-specific antibodies such as a
IgA, IgM, IgG or total antibodies using immunoassays. Serology is not interchangeable with and does not
replace nuclear acid amplification tests or antigen tests for detection of SARS-CoV-2. Serology should not be
used as a standalone diagnostic test to identify acute cases in clinical care or for contact tracing purposes.

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[Slide #19] When the clinical suspicion for SARS-CoV-2 infection is high, but is not supported by adequate
and repeated nucleic acid amplification testing, serology can support in determining retrospective SARS-CoV-
2 infection. In this situation, a serum sample taken in the acute phase as well as a sample at a later time point
generally 2 to 4 weeks later should be obtained from the patients.

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[Slide #20] Interpretation of serology test results should be made by an expert. Interpretations are dependent
on several factors such as timing of symptoms, clinical morbidity, the epidemiology, and prevalence within the
setting, the type of test used, the validation method, and the reliability of the results. Detection of antibodies to
SARS-CoV-2 does not guarantee that they are neutralizing antibodies or that they offer protective immunity.

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[Slide #21] There are also several non-specific laboratory abnormalities that have been described in COVID-
19 infections, such as leukocytosis or elevated white blood cells. Lymphopenia or decrease white blood cells,
increased neutrophils, elevated aminotransaminase levels. Decreased hypoalbuminemia levels, elevated
lactate dehydrogenase levels. Elevated inflammatory markers such as the C reactive protein, erythrocyte
sedimentation rate, IL-6, ferritin, D-Dimer, hyperglycemia, prolonged thrombin time, and elevated activated
partial thromboplastin time. Elevated fibrinogen and increase fibrin degradation products. An elevated D-Dimer
created than 1 μg/ml has been determined to be an independent risk factor for mortality.

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[Slide #22] When SARS-CoV-2 is expected, do not forget to also consider other co-infections. Remember, a
positive test for SARS-CoV-2 does not rule out co-infection with another pathogen. Another for patient
symptoms. In an acute setting, collect additional specimens to aid further in laboratory evaluation. For example,
in high prevalence of tuberculosis considers sputum for bacteriology. Specimen should also be taken from
other sites that may be infected, such as urine, cerebrospinal fluid, stool, pleural fluid, peritoneal fluid and can
yield pathogens, as clinically indicated. For patient's sepsis, two sets of blood cultures for bacteriology from
two different sites, where possible, should be obtained. Please note that SARS-CoV-2 has not be shown to be
transmitted by blood.

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OpenWHO: Clinical Management of patients with COVID-19: Investigations and care of mild, moderate and sever
disease

[Slide #23] So this concludes the module on Laboratory Considerations for COVID-19, part of the Clinical
Management Course series. Listed here for you,some additional resources on this topic. Thank you very much
for listening.