Coronavirus disease (COVID-19) technical guidance by WHO Laboratory testing for COVID-19 | Page 3

Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases
Infectious Substances 2019-2020”(22) and “WHO
interim guidance for laboratory biosafety related to
2019-nCoV”(18).
whole genome of the virus as long as the
sequence target is larger or different from
the amplicon probed in the NAAT assay
used.
Ensure good communication with the laboratory
and provide needed information
Alerting the laboratory before sending specimens
encourages proper and timely processing of samples
and timely reporting. Specimens should be correctly
labelled and accompanied by a diagnostic request
form (template provided in Annex I).
When there are discordant results, the patient should
be resampled and, if appropriate, sequencing of the
virus from the original specimen or of an amplicon
generated from an appropriate NAAT assay,
different from the NAAT assay initially used, should
be obtained to provide a reliable test result.
Laboratories are urged to seek confirmation of any
surprising results in an international reference
laboratory.
4. Laboratory testing for COVID-19 virus
Laboratory confirmed case by NAAT in areas
with established COVID-19 virus circulation
In areas where COVID-19 virus is widely spread a
simpler algorithm might be adopted in which for
example screening by rRT-PCR of a single
discriminatory target is considered sufficient.
Laboratories undertaking testing for COVID-19
virus should adhere strictly to appropriate biosafety
practices.
Nucleic acid amplification tests (NAAT) for
COVID-19 virus
Routine confirmation of cases of COVID-19 is
based on detection of unique sequences of virus
RNA by NAAT such as real-time reverse-
transcription polymerase chain reaction (rRT-PCR)
with confirmation by nucleic acid sequencing when
necessary. The viral genes targeted so far include the
N, E, S and RdRP genes. Examples of protocols used
may be found here. RNA extraction should be done
in a biosafety cabinet in a BSL-2 or equivalent
facility. Heat treatment of samples prior to RNA
extraction is not recommended.
One or more negative results do not rule out the
possibility of COVID-19 virus infection. A number
of factors could lead to a negative result in an
infected individual, including:
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Laboratory confirmation of cases by NAAT in
areas with no known COVID-19 virus circulation
To consider a case as laboratory-confirmed by
NAAT in an area with no COVID-19 virus
circulation, one of the following conditions need to
be met:
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poor quality of the specimen, containing
little patient material (as a control, consider
determining whether there is adequate
human DNA in the sample by including a
human target in the PCR testing)
the specimen was collected late or very
early in the infection
the specimen was not handled and shipped
appropriately
technical reasons inherent in the test, e.g.
virus mutation or PCR inhibition.
If a negative result is obtained from a patient with a
high index of suspicion for COVID-19 virus
infection, particularly when only upper respiratory
tract specimens were collected, additional
specimens, including from the lower respiratory
tract if possible, should be collected and tested.
A positive NAAT result for at least two
different targets on the COVID-19 virus
genome, of which at least one target is
preferably specific for COVID-19 virus
using a validated assay (as at present no
other SARS-like coronaviruses are
circulating in the human population it can
be debated whether it has to be COVID-19
or SARS-like coronavirus specific); OR
One positive NAAT result for the presence
of betacoronavirus, and COVID-19 virus
further identified by sequencing partial or
Each NAAT run should include both external and
internal controls, and laboratories are encouraged to
participate in external quality assessment schemes
when they become available. It is also recommended
to laboratories who order their own primers and
probes to perform entry testing/validation on
functionality and potential contaminants.
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