Diagnostic testing and screening for SARS-CoV-2

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Diagnostic specimens

Samples for diagnostic tests for SARS-CoV-2 can be taken from the upper (nasopharyngeal/oropharyngeal swabs, nasal aspirate, nasal wash or saliva) or lower respiratory tract (sputum or tracheal aspirate or bronchoalveolar lavage - BAL). Data comparing the accuracy of RT-PCR testing suggest that test sensitivity may vary by type of specimen.

One study suggested that viral RNA levels are higher and RNA is more frequently detected in nasal specimens as compared to oral specimens, however this finding was based on a small number of nasal swabs tested [1]. A COVID-19 investigation team in the US comparing 117 pairs of nasopharyngeal and oropharyngeal specimens from 12 patients simultaneously, found that 32 pairs were discordant with one test positive and the other negative: the nasopharyngeal specimen tested positive in 66% of those pairs compared with 34% for the oropharyngeal specimen [2]. Another study did not show higher viral RNA levels in nasopharyngeal compared with oropharyngeal specimens [3].  

When comparing different types of specimens, preliminary results from a pre-print article not yet peer-reviewed showed that the most accurate sample for the diagnosis of SARS-CoV-2 was sputum, followed by nasal swabs and throat swabs [4]. A meta-analysis of saliva testing studies found 91% (95%CI = 80%-99%) sensitivity for saliva tests and 98% (95%CI 89%-100%) sensitivity for nasopharyngeal swab tests in previously confirmed COVID-19 infected patients, with moderate heterogeneity among studies [5]. Another study showed that saliva was the most appropriate sample for diagnosis of SARS-CoV-2[6]. Saliva offers a non-invasive specimen that can also be considered for self-sampling. In a situation where a nasopharyngeal or other above mentioned specimen is not acceptable, saliva could be considered as an alternative specimen.

The combination of nasopharyngeal/oropharyngeal swab samples proved more sensitive for diagnosis of SARS-CoV-2 compared to nasopharyngeal swab only in three different studies [7] [8,9].

Assay types

There are three main types of detection assays relevant for COVID-19 diagnostic testing and screening, based on the target that is being detected:

  • Nucleic acid tests detect the presence of viral RNA. Typically, these use an amplification step based on RT-PCR.
  • Antigen tests detect the presence of a viral antigen, typically part of a surface protein.
  • Antibody tests detect the presence of antibodies generated against SARS-CoV-2. The three most used assays are enzyme-linked immunosorbent assays (ELISA), chemoluminescence assays (CLIA) and lateral flow assays (LFA). In addition, virus neutralisation tests are used, which can specifically detect neutralising antibodies, but this is mainly used for assay validation and research. Preliminary reports on ELISA assays have shown good correlation of antibody titration results with virus-neutralising antibodies [116,160].

Apart from these main detection assays, whole genome sequencing can also be performed to determine the sequence of the SARS-CoV-2 virus in a sample, with possible quasi-species variants [10].

In-house tests versus in vitro diagnostic medical devices

In-house tests are non-commercial in vitro test methods performed in laboratories, following a scientifically published protocol after internal performance verification, in accordance with their quality assurance system based on international clinical laboratory quality standards. In-house real-time reverse transcription polymerase chain reaction (RT-PCR) test methods targeting SARS-CoV-2 viral RNA are the gold standard in vitro methods for diagnosing suspected cases of COVID-19. These PCR tests can be automated by using robotic molecular platforms for high-throughput batch processing of clinical specimens.

In vitro diagnostic medical devices (IVDs) are commercial kits or systems that must be affixed the CE-IVD label to be placed on the EU market. The IVD Directive 98/79/EC [11] defines an ‘in vitro diagnostic medical device’ as any medical device which is a reagent, reagent product, calibrator, control material, kit, instrument, apparatus, equipment, or system, whether used alone or in combination, intended by the manufacturer to be used in vitro for the examination of specimens, including blood and tissue donations, derived from the human body, solely or principally for the purpose of providing information, in the COVID-19 context, concerning a physiological or pathological state, or to monitor therapeutic measures. The ‘intended purpose’ of IVD means the use for which the device is intended according to the data supplied by the manufacturer on the labelling, in the instructions for use and/or in promotional materials. According to IVD Directive 98/79/EC Article 9 [11] on conformity assessment procedures, for COVID-19 diagnostic devices that are not intended for use as self-tests, the manufacturer shall, in order to affix the CE marking, draw up the EC declaration of conformity required before placing the devices on the market. This is a self-declaration procedure based on satisfying essential safety and performance requirements listed in the Directive and specifications of the device performance characteristics, stated by the manufacturer. In case of self-tests, involvement of a third-party conformity assessment body is necessary. The performance of point-of-care and rapid diagnostic test devices put on the EU market with CE label may vary in the laboratory in comparison to the performance study of the manufacturer done for the purposes of CE-marking. There are several hundreds of CE-marked IVDs for COVID-19 virus or antibody detection: independent evaluation of their diagnostic accuracy is ongoing.

Intended use

Diagnostic testing for COVID-19 by viral RNA or protein detection in respiratory specimens supports decision making for clinical, infection control or public health management. SARS-CoV-2 detection for diagnosis of patients with COVID-19-like symptoms is essential for patient care, triage and isolation in healthcare facilities.

SARS-CoV-2 detection can also be used for screening close contacts for asymptomatic infection and disease as part of contact tracing or outbreak investigations. Testing is also used to screen for infection in crucial target groups like healthcare and social workers as part of local surveillance programmes. This is especially important for prevention and early control of viral transmission to vulnerable persons living in closed institutions such as long-term care facilities.

Apart from real-time use for medical or public health case management and transmission control, virus detection tests are used for policy-oriented surveillance purposes to monitor the epidemiologic situation in terms of incidence and prevalence of infection and disease. This use includes prevalence surveys and sentinel surveillance programmes in the community, primary care or hospital care patient populations.

Antibody tests currently have limited diagnostic use. They can be used as a complement to the virus detection tests for patients presenting late after symptoms onset to healthcare facilities and where virus detection tests are negative despite strong indications of infection. In addition, they can potentially be used for informing the decision on discharge of patients who recovered from SARS-CoV-2 infection but remain RNA-positive by RT-PCR for a long time after symptoms have subsided. The degree of protective immunity conferred by or correlated with the antibodies detected in subjects with past SARS-CoV-2 infection is still under investigation. Once this is clarified, such antibody tests could be, together with the direct virus detection, an essential tool in de-escalation strategies. Currently antibody tests are used for sero-epidemiological surveys and studies.

Rapidity and point of testing

Two other important aspects of detection assays are their rapidity and ease of use. The common technical specifications for in vitro diagnostic medical devices (IVDs, Commission Decision 2002/364/EC) [12] define rapid tests as qualitative or semi-quantitative IVDs, used once or in a small series, which involve non-automated procedures and have been designed to give a fast result. Typically, rapid tests can be performed under 30 minutes.

Tests that can be performed at the point-of-care, by less specialised personnel, are called point of care tests (POCTs). POCTs are normally rapid tests as well.

When rapid antigen tests are well-validated, they may be considered for the rapid diagnosis of infected patients. However, these tests tend to have lower sensitivity than RT-PCR, and therefore a negative rapid test may not be able to rule out infection. They may be useful during an ongoing outbreak, when timely access to sensitive molecular testing is unavailable, but a negative result should be interpreted by a healthcare professional with caution and based on clinical judgement.

Diagnostic accuracy and validation

The Commission has published a working document which proposes a tentative definition of COVID-19 diagnostic test performance criteria and has reviewed publically available data on the performance of CE-marked commercial IVD tests [13]. These criteria include analytical sensitivity, analytical specificity, clinical sensitivity and clinical specificity. As a follow-up of this document, the European Commission is collating, in a searchable database, the manufacturer data of CE-marked commercial IVD tests and reviewing in-house laboratory-developed tests with performance data in scientific publications [14].

A large number of commercial detection assays for SARS-CoV-2 RNA or antigen and serological assays for SARS-CoV-2 specific antibodies are being placed on the market or are potentially exportable to EU countries with CE-IVD marking. However, information on their clinical performance is still limited and further scientific validation of their diagnostic accuracy, i.e. clinical sensitivity and specificity, is a priority. This must be performed in real-life prospective cohort studies of the intend-to-test patient or general population. The Foundation for Innovative New Diagnostics (FIND), WHO Collaborating Centre for Laboratory Strengthening and Diagnostic Technology Evaluation, has received over 600 manufacturers’ submissions for IVDs, and has been performing independent clinical evaluations in a few FIND collaborating hospitals on a subset of them. ECDC has been collecting clinical performance data on commercial assays since 1 April from Member States’ laboratories willing to share such information with each other.

WHO experts, through the Emergency Use Listing procedure (EUL), shortlist molecular detection assays based on manufacturers’ data as suitable for emergency procurement prior to full clinical validation and pre-qualification [15].

Biosafety

On 3 June 2020 the Commission adopted Directive (EU) 2020/739 [16] updating the Biological Agents Directive 2000/54/EC with classification of SARS-CoV-2 as risk group 3 in the Annex III- list of biological agents. In line with Article 16(1)(c), and in accordance with WHO interim recommendations on laboratory biosafety for COVID-19 laboratory procedures, non-propagative diagnostic laboratory work involving SARS-CoV-2 should be conducted at a facility using procedures equivalent to at least containment level 2. Propagative work (virus culture, neutralisation assays) involving infectious SARS-CoV-2 should be conducted at a containment level 3 laboratory with air pressure negative to atmosphere.

Testing capacity and methodologies

As part of the Joint European Roadmap towards lifting COVID-19 containment measures, the European Commission has issued Guidelines on COVID-19 in vitro diagnostic tests and their performance [17]. These guidelines assess both what information different types of tests can deliver for medical and public health decision-making and how to validate that the test performance is fit for purpose. To foster scaling up of the testing capacity and ensure adequate quality of tests across the EU, the Commission has undertaken a number of actions including: 

  • an assessment of common approaches in national testing strategies;
  • the discussion of best practices and development of guidance on performance evaluation and conformity assessment of tests;
  • the provision of reference materials and common methods for the comparison of devices; 
  • the sharing of information on the performance of tests; 
  • additional dialogue with industry and national competent authorities;
  • support in the fight against counterfeit devices;
  • coordination of supply and demand; 
  • ensuring the fair distribution of laboratory supplies between Member States.

In this context, ECDC contributes to capacity building and test validation efforts by mobilising the knowledge and experience from Member States within the European networks of public health experts and reference laboratories. ECDC coordinates a COVID-19/SARS-CoV-2 network which includes laboratory experts and discusses key laboratory aspects on a regular basis within the network. In close collaboration with WHO and WHO referral laboratories, ECDC is organising external quality assessment exercises and facilitating exchange of information on test performance between Member States’ public health laboratories from the COVID-19 laboratory network.

Despite shortages of consumables in the past, testing capacity for virus detection has rapidly expanded in EU/EEA countries by the roll-out of PCR-based diagnostics from central public health laboratories to regional and local diagnostic laboratories and the use of high-throughput automated molecular testing platforms. However, additional capacity for much larger scale testing with rapid commercial tests, once such tests are validated to have adequate performance for infection detection, will most likely be necessary to fully meet the operational needs for COVID-19 control in the forthcoming months.

 

References