Key messages:
* Transcription-mediated amplification (TMA) tests and commercially available reverse transcription polymerase chain reaction (RT-PCR) tests specifically designed to omit/adapt RNA extraction/purification appear accurate enough to replace the method of RT-PCR with prior RNA (a molecule essential for most biological functions) extraction/purification for identifying people infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially when the alternative would be not having access to testing. However, these findings need to be interpreted and used with caution because of several limitations in the evidence.
* The accuracy of other types of laboratory-based molecular alternatives to RT-PCR is below World Health Organisation (WHO) recommended standards or lacks a sufficient volume of evidence to draw reliable conclusions.
*The extrapolation of accuracy data from the evaluated tests to any other test brand(s) using the same techniques will be challenging since main categories are overrepresented by highly accurate individual test brands. Further evaluation of these tests in real clinical practice scenarios is needed.
What are alternative laboratory-based molecular assays for identifying people infected by SARS-CoV-2?
Laboratory-based molecular assays (tests) aim to confirm or rule out SARS-CoV-2 infection in people. Alternatives to RT-PCR assays have been developed to minimise the steps needed to process samples in the laboratory or use fewer resources to obtain the same valid results. For this review, we focus on tests commercially developed by a manufacturer, providing instructions for their professional use.
Why is this question important?
People with suspected SARS-CoV-2 infection should know if they are infected to self-isolate, receive treatment, and inform close contacts. Failure to detect SARS-CoV-2 when it is present risks spreading infection and results in missed opportunities for prevention. Diagnosing SARS-CoV-2 infection when it is not present may lead to unnecessary self-isolation and testing of close contacts. Currently, testing to confirm SARS-CoV-2 infection relies on a laboratory molecular test called RT-PCR. This requires specialist equipment and can take 24 hours to produce a result. If assays that take less time or use readily available resources were sufficiently accurate to replace RT-PCR testing, this could increase the number of tests that could be done, allow faster diagnosis and enable people to take appropriate action more quickly.
What did we want to find out?
We wanted to know whether commercially developed, alternative laboratory-based molecular tests are accurate enough to diagnose SARS-CoV-2 infection compared to RT-PCR.
What did we do?
We looked for studies investigating these tests in people (or samples) that were also tested for SARS-CoV-2 infection using RT-PCR which modified RT-PCR by removing or altering components of this testing process.
What did we find?
We included 105 evaluations evaluating alternatives to RT-PCR in the review. The main results are based on 74,753 samples, and SARS-CoV-2 infection was confirmed in 7517 of these samples. Most evaluations (27/105) concerned various test types specifically designed to omit/adapt RNA extraction/purification. Twenty-four of 105 evaluations concerned various test types operating at one temperature (isothermal tests) rather than cycling through different temperatures. Twenty-four of 105 evaluations were concerned with various test types that combined these two alternative approaches.
Main results
Only tests specifically designed to omit/adapt RNA extraction/purification and TMA assays with RNA extraction (a type of isothermal test) met WHO-acceptable standards for confirming and ruling out SARS-CoV-2. For illustration, the results of these studies indicate that in a group of 1000 people, 50 of whom (5%) actually have SARS-CoV-2:
* For tests specifically designed to omit/adapt RNA extraction/purification:
-51 people would test positive for SARS-CoV-2. Of these, three people (6%) would not have SARS-CoV-2 (false-positive result).
-949 people would test negative for SARS-CoV-2. Of these, two people (0.2%) would actually have SARS-CoV-2 (false-negative result)
*For TMA tests with RNA extraction:
-55 people would test positive for SARS-CoV-2. Of these, six people (10%) would not have SARS-CoV-2 (false-positive result).
-945 people would test negative for SARS-CoV-2. Of these, one person (0.1%) would actually have SARS-CoV-2 (false-negative result)
What are the limitations of the evidence?
Most included studies did not provide information on participants who underwent testing, such as whether they had symptoms or were close contacts of a SARS-CoV-2 case. We also had concerns about the method of recruiting participants for the majority of included studies, which may have resulted in an overestimation of the accuracy of these methods. In addition, the method used to determine whether individuals did not have SARS-CoV-2 infection was not considered reliable for most studies, which may have resulted in underestimating the accuracy of these methods. Some studies did not follow the manufacturers' instructions for using the test. Results from different test brands using the same technique varied.
What does this mean?
The studies included in this review suggest that two laboratory-based molecular tests might be accurate enough to replace or supplement RT-PCR tests with prior RNA extraction/purification for the diagnosis of SARS-CoV-2 infection: RT-PCR tests developed to omit or adapt RNA extraction or purification and TMA isothermal tests (retaining an RNA extraction step). Other alternative tests to RT-PCR were assessed just by a few studies of limited methodological quality, and their performance should be evaluated by additional studies. Furthermore, data in these studies mostly relied on samples acquired in the past (compared to current or future samples), thus further evaluation of these tests in real clinical practice scenarios is required. Also, decisions on the optimal test for a specific setting will be driven not only by diagnostic accuracy, but also by other factors such as test complexity, time to result, acceptability to those being tested, and the setting in which the tests are to be used.
How up‐to‐date is this review?
This review includes evidence published up to 31 October 2022.
Alternative laboratory-based molecular tests aim to enhance testing capacity in different ways, such as reducing the time, steps and resources needed to obtain valid results. Several index test technologies with these potential advantages have not been evaluated or have been assessed by only a few studies of limited methodological quality, so the performance of these kits was undetermined.
Only two index test categories with enough evaluations for meta-analysis fulfil the WHO set of acceptable accuracy standards for SARS-CoV-2 nucleic acid tests: RT-PCR assays designed to omit/adapt RNA extraction/purification and TMA assays. These assays might prove to be suitable alternatives to RT-PCR for identifying people infected by SARS-CoV-2, especially when the alternative would be not having access to testing. However, these findings need to be interpreted and used with caution because of several limitations in the evidence, including reliance on retrospective samples without information about the symptom status of participants and the timing of assessment. No extrapolation of found accuracy data for these two alternatives to any test brands using the same techniques can be made as, for both groups, one test brand with high accuracy was overrepresented with 21/26 and 12/14 included studies, respectively. Although we used a comprehensive search and had broad eligibility criteria to include a wide range of tests that could be alternatives to RT-PCR methods, further research is needed to assess the performance of alternative COVID-19 tests and their role in pandemic management.
Diagnosing people with a SARS-CoV-2 infection played a critical role in managing the COVID-19 pandemic and remains a priority for the transition to long-term management of COVID-19. Initial shortages of extraction and reverse transcription polymerase chain reaction (RT-PCR) reagents impaired the desired upscaling of testing in many countries, which led to the search for alternatives to RNA extraction/purification and RT-PCR testing. Reference standard methods for diagnosing the presence of SARS-CoV-2 infection rely primarily on real-time reverse transcription-polymerase chain reaction (RT-PCR). Alternatives to RT-PCR could, if sufficiently accurate, have a positive impact by expanding the range of diagnostic tools available for the timely identification of people infected by SARS-CoV-2, access to testing and the use of resources.
To assess the diagnostic accuracy of alternative (to RT-PCR assays) laboratory-based molecular tests for diagnosing SARS-CoV-2 infection.
We searched the COVID‐19 Open Access Project living evidence database from the University of Bern until 30 September 2020 and the WHO COVID-19 Research Database until 31 October 2022. We did not apply language restrictions.
We included studies of people with suspected or known SARS‐CoV‐2 infection, or where tests were used to screen for infection, and studies evaluating commercially developed laboratory-based molecular tests for the diagnosis of SARS-CoV-2 infection considered as alternatives to RT-PCR testing. We also included all reference standards to define the presence or absence of SARS‐CoV‐2, including RT‐PCR tests and established clinical diagnostic criteria.
Two authors independently screened studies and resolved disagreements by discussing them with a third author. Two authors independently extracted data and assessed the risk of bias and applicability of the studies using the QUADAS‐2 tool. We presented sensitivity and specificity, with 95% confidence intervals (CIs), for each test using paired forest plots and summarised results using average sensitivity and specificity using a bivariate random‐effects meta‐analysis. We illustrated the findings per index test category and assay brand compared to the WHO's acceptable sensitivity and specificity threshold for diagnosing SARS-CoV-2 infection using nucleic acid tests.
We included data from 64 studies reporting 94 cohorts of participants and 105 index test evaluations, with 74,753 samples and 7517 confirmed SARS-CoV-2 cases. We did not identify any published or preprint reports of accuracy for a considerable number of commercially produced NAAT assays. Most cohorts were judged at unclear or high risk of bias in more than three QUADAS-2 domains. Around half of the cohorts were considered at high risk of selection bias because of recruitment based on COVID status. Three quarters of 94 cohorts were at high risk of bias in the reference standard domain because of reliance on a single RT-PCR result to determine the absence of SARS-CoV-2 infection or were at unclear risk of bias due to a lack of clarity about the time interval between the index test assessment and the reference standard, the number of missing results, or the absence of a participant flow diagram.
For index tests categories with four or more evaluations and when summary estimations were possible, we found that: a) For RT-PCR assays designed to omit/adapt RNA extraction/purification, the average sensitivity was 95.1% (95% CI 91.1% to 97.3%), and the average specificity was 99.7% (95% CI 98.5% to 99.9%; based on 27 evaluations, 2834 samples and 1178 SARS-CoV-2 cases); b) For RT-LAMP assays, the average sensitivity was 88.4% (95% CI 83.1% to 92.2%), and the average specificity was 99.7% (95% CI 98.7% to 99.9%; 24 evaluations, 29,496 samples and 2255 SARS-CoV-2 cases); c) for TMA assays, the average sensitivity was 97.6% (95% CI 95.2% to 98.8%), and the average specificity was 99.4% (95% CI 94.9% to 99.9%; 14 evaluations, 2196 samples and 942 SARS-CoV-2 cases); d) for digital PCR assays, the average sensitivity was 98.5% (95% CI 95.2% to 99.5%), and the average specificity was 91.4% (95% CI 60.4% to 98.7%; five evaluations, 703 samples and 354 SARS-CoV-2 cases); e) for RT-LAMP assays omitting/adapting RNA extraction, the average sensitivity was 73.1% (95% CI 58.4% to 84%), and the average specificity was 100% (95% CI 98% to 100%; 24 evaluations, 14,342 samples and 1502 SARS-CoV-2 cases).
Only two index test categories fulfil the WHO-acceptable sensitivity and specificity requirements for SARS-CoV-2 nucleic acid tests: RT-PCR assays designed to omit/adapt RNA extraction/purification and TMA assays. In addition, WHO-acceptable performance criteria were met for two assays out of 35 when tests were used according to manufacturer instructions. At 5% prevalence using a cohort of 1000 people suspected of SARS-CoV-2 infection, the positive predictive value of RT-PCR assays omitting/adapting RNA extraction/purification will be 94%, with three in 51 positive results being false positives, and around two missed cases. For TMA assays, the positive predictive value of RT-PCR assays will be 89%, with 6 in 55 positive results being false positives, and around one missed case.