This article provides interim guidance to laboratories and stakeholders involved in COVID-19 virus laboratory testing of patients.
Laboratory Testing Guiding Principles
The decision to test should be based on clinical and epidemiological factors and linked to an assessment of the likelihood of infection. PCR testing of asymptomatic or mildly symptomatic contacts can be considered in the
assessment of individuals who have had contact with a COVID-19 case. Screening protocols should be adapted to the local situation.
Rapid collection and testing of appropriate specimens from patients meeting the suspected case definition for COVID-19 is a priority for clinical management and outbreak control and should be guided by a laboratory expert. Suspected cases should be screened for the virus with nucleic acid
amplification tests (NAAT), such as RT-PCR.
If case management requires, patients should be tested for other respiratory pathogens using routine laboratory procedures, as recommended in local management guidelines for community-acquired pneumonia. Additional testing in the laboratory should not delay testing for COVID-19. As co-infections can occur, all patients that meet the suspected case definition should be tested for COVID-19 virus regardless of whether another respiratory pathogen is found.
In an early study in Wuhan, the mean incubation period for COVID-19 was 5.2 days among 425 cases, though it varies widely between individuals. Virus shedding patterns are not yet well understood and further investigations are needed to better understand the timing, compartmentalization, and quantity of viral shedding to inform optimal specimen collection. Although respiratory samples have the greatest
yield, the virus can be detected in other specimens, including stool and blood. Local guidelines on informed consent should be followed for specimen collection, testing, and potentially future research.
Specimen Collection and Shipment
Safety procedures during specimen collection
Ensure that adequate standard operating procedures (SOPs) are in use and that staff is trained for appropriate specimen collection, storage, packaging, and transport. All specimens collected for laboratory investigations should be regarded as potentially infectious.
Ensure that health care workers who collect specimens adhere rigorously to infection prevention and control guidelines.
Specimens to be collected
At a minimum, respiratory material should be collected:
upper respiratory specimens: nasopharyngeal and oropharyngeal swab or wash in ambulatory patients
and/or lower respiratory specimens: sputum (if produced) and/or endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory disease. (Note high risk of aerosolization; adhere strictly to infection prevention and control procedures).
Additional clinical specimens may be collected as COVID-19 virus has been detected in blood and stool, as had the coronaviruses responsible for SARS and MERS. The duration and frequency of shedding of the COVID-19 virus in stool and potentially in urine is unknown. In the case of patients who are deceased, consider autopsy material including lung tissue. In surviving patients, paired serum (acute and convalescent) can be useful to retrospectively define cases as serological assays become available.
Packaging And Shipment Of Clinical Specimens
Specimens for virus detection should reach the laboratory as soon as possible after collection. Correct handling of specimens during the transportation is essential. Specimens that can be delivered promptly to the laboratory can be stored and shipped at 2-8°C.
When there is likely to be a delay in specimens reaching the laboratory, the use of the viral transport medium is strongly recommended. Specimens may be frozen to – 20°C or ideally -70°C and shipped on dry ice if further
delays are expected (see Table 2). It is important to avoid repeated freezing and thawing of specimens.
Transport of specimens within national borders should comply with applicable national regulations. International transport of potentially COVID-19 virus-containing samples should follow the UN Model Regulations, and any other applicable regulations depending on the mode of transport being used.
Ensure Good Communication With the Laboratory And Provide NeededIinformation.
Alerting the laboratory before sending specimens (COVID-19) encourages proper and timely processing of samples and timely reporting after testing. Specimens should be correctly labeled and accompanied by a diagnostic request form.
Laboratory testing for COVID-19 virus
Laboratories undertaking to test for COVID-19 virus should adhere strictly to appropriate biosafety practices.
Nucleic Acid Amplification Tests (NAAT) For COVID-19 Virus.
Routine laboratory confirmation testing of cases of COVID-19 is based on the 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 before RNA extraction is not recommended.
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:
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-1 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 must be COVID-19 or SARS-like coronavirus specific);
OR
One positive NAAT result for the presence of beta coronavirus, and COVID-19 virus further identified by sequencing partial or whole genome of
the virus as long as the sequence target is larger or different from the amplicon probed in the NAAT assay used.
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.
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.
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:
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.
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 that order their own primers and probes to perform entry
testing/validation on functionality and potential contaminants.
Serological testing
Serological surveys can aid investigation of an ongoing outbreak and retrospective assessment of the attack rate or extent of an outbreak. In cases where NAAT assays are negative and there is a strong epidemiological link to COVID-19 infection, paired serum samples (in the acute and convalescent phase) could support diagnosis once validated serology tests are available. Serum samples can be stored for these purposes.
Cross-reactivity to other coronaviruses can be challenging, but commercial and non-commercial serological tests are currently under development. Some studies with COVID-19 serological data on clinical samples have been published.
Viral Sequencing
In addition to providing confirmation of the presence of the virus, regular sequencing of a percentage of specimens from clinical cases can be useful to monitor for viral genome mutations that might affect the performance of medical countermeasures, including diagnostic tests. Virus whole-genome sequencing can also inform molecular epidemiology studies. Many public-access databases for deposition of genetic sequence data are available, including GISAID, which is intended to protect the rights of the submitting
party.
Viral culture
Virus isolation is not recommended as a routine diagnostic procedure.
Reporting Of Cases And Test Results
Laboratories should follow national reporting requirements. In general, all test results, positive or negative, should be immediately reported to national authorities. States Parties to the IHR are reminded of their obligations to share with WHO relevant public health information for events for which they notified WHO, using the decision instrument in Annex 1 of the IHR (2005).
Research Toward Improved Detection Of COVID-19 Virus.
Many aspects of the virus and disease are still not understood. A better understanding will be needed to provide improved guidance. For example:
Viral dynamics: optimal timing and type of clinical material to sample for molecular testing-
Dynamic of immunological response
Disease severity in various populations, e.g. by age.
The relationship between viral concentration and disease severity.
The duration of shedding, and relation to clinical picture (e.g. clinical recovery occurs with viral clearing, or shedding persists despite clinical
improvement).
Development and validation of useful serological assays.
Comparative studies of available molecular and serological assays.
The optimal percentage of positive cases that requires sequencing to monitor mutations that might affect the performance of molecular tests.
WHO encourages the sharing of data to better understand and thus manage the OVID-19 outbreak, and to develop countermeasures.
| Description | Test | Type | Timing |
| Patient | NAAT | Lower respiratory tract sputum aspirate lavage Upper respiratory tract nasopharyngeal and oropharyngeal swabs nasopharyngeal wash/nasopharyngeal aspirate. | Collect on presentation. Possibly repeated sampling to monitor clearance. Further research needed to determine effectiveness and reliability of repeated sampling. |
| Patient | Serology | Consider stools, whole blood, urine, and if diseased, material from autopsy Serum for serological testing once validated and available. | Paired samples are necessary for confirmation with the initial sample collected in the first week of illness and the second ideally collected 2-4 weeks later (optimal timing for convalescent sample needs to be established). |
| Specimen Type | Collection Materials | Storage temperature until testing in-country laboratory | Recommended temperature for shipment according to expected shipment time |
| Nasopharyngeal and oropharyngeal swab | Dacron or polyester flocked swabs | 2-8 °C | 2-8 °C if ≤5 days –70 °C (dry ice) if >5 days |
| Bronchoalveolar lavage | Sterile Container | 2-8 °C | 2-8 °C if ≤2 days –70 °C (dry ice) if >2 days |
| (Endo)tracheal aspirate, nasopharyngeal or nasal wash/aspirate | Sterile Container | 2-8 °C | 2-8 °C if ≤2 days –70 °C (dry ice) if >2 days |
| Sputum | Sterile Container | 2-8 °C | 2-8 °C if ≤2 days –70 °C (dry ice) if >2 days |