An overview of the most important preanalytical factors influencing the clinical performance of SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs)

Patient’s age

Although it is not straightforwardly clear why the clinical performance of SARS-CoV-2 RDTs may be dependent upon patient’s age, several lines of evidence converge to confirm that the accuracy of this type of testing seems lower at certain ages, especially using self-collected specimens. An interesting report published by the CDC COVID-19 Response Team demonstrated that the accuracy of self-collected specimens was lower in the age range between 30 and 49 years (i.e., 0.45–0.67 sensitivity) and ≥60 years (i.e., 0.68 sensitivity) compared to other age ranges (i.e., sensitivity ≥0.85) [15]. A reduced diagnostic sensitivity has also been clearly reported in children compared to adults (i.e., 0.64 vs. 0.73) by the Cochrane COVID-19 Diagnostic Test Accuracy Group [13]. In general, no major differences in test accuracy has been found between sexes [16].

Presence and duration of symptoms

The presence, duration and type of symptoms are variables that are widely known to influence the diagnostic accuracy of SARS-CoV-2 molecular tests [17], but may have a substantial, even greater, impact also on SARS-CoV-2 Ag-RDTs. One of the possible (though not exclusive) explanation, is that the viral load varies widely over the course of a SARS-CoV-2 acute infection. Specifically, the incubation time of the SARS-CoV-2 Omicron lineages is now significantly faster (i.e., around 3.0–3.8 days) compared to the former variants (i.e., between 4 and 5 days) [18, 19], which seems to be also accompanied by quicker decrease of viral load in the upper respiratory tract, faster viral clearance and more rapid resolution of symptoms (i.e., typically between 2 and 6 days in people infected by Omicron lineages compared to 2–14 days in those infected by the Delta variant) [20, 21].

It is hence not surprising that the diagnostic accuracy (more specifically, the diagnostic sensitivity) of SARS-CoV-2 Ag-RDTs would be higher (i) in symptomatic compared to asymptomatic subjects, (ii) in those with shorter onset of symptoms, as well as in (iii) patients with symptoms prevailing in the upper respiratory tract, where the diagnostic samples are taken. The meta-analysis published by the Cochrane COVID-19 Diagnostic Test Accuracy Group highlighted that the pooled diagnostic sensitivity of SARS-CoV-2 Ag-RDTs is 0.73 (95% CI, 0.69–0.79) in symptomatic subjects, compared to 0.54 (95% CI, 0.48–0.62) in those without symptoms [13]. Tests targeting contacts or cohorts specifically referred for diagnostic testing yielded also higher diagnostic sensitivity (i.e., 0.64; 95% CI; 0.55–0.73) compared to random testing of asymptomatic people (i.e., 0.50; 95% CI; 0.42–0.57). In keeping with this evidence, the diagnostic sensitivity obtained in healthcare facilities (i.e., 0.61; 95% CI; 0.54–0.68) was found to be superior to that observed during mass (0.45; 95% CI; 0.36–0.54) or school (i.e., 0.48; 95% CI; 0.38–0.58) screening programs. The dependency of the diagnostic sensitivity of SARS-CoV-2 Ag-RDTs from clinical disease has been confirmed in other meta-analyses. More specifically, Brümmer et al. reported a diagnostic sensitivity of 0.76 (95% CI, 0.73–0.79) in symptomatic subjects, compared to 0.57 (95% CI, 0.51–0.62) in those who did not report symptoms [22].

As then concerns the time of symptom onset, as predictable, the Cochrane COVID-19 Diagnostic Test Accuracy Group has evidenced that the diagnostic sensitivity of SARS-CoV-2 Ag-RDTs is nearly twofold higher in those who developed symptoms <1 week (i.e., 0.81; 95% CI, 0.88–0.84) compared to those who complained for symptoms started for over one week (i.e., 0.49; 95% CI, 0.38–0.60) [13]. Brümmer et al. also estimated a diagnostic sensitivity of 0.82 (95% CI, 0.78–0.85) in patients with symptom onset <1 week, compared to 0.52 (95% CI, 0.41–0.62) in those who reported onset of symptoms for ≥1 week [22]. In the meta-analysis published by Xie et al., the diagnostic sensitivity of SARS-CoV-2 Ag-RDTs gradually decreased in parallel with the time passed since onset of the symptoms, being as high as 0.91 (95% CI, 0.83–0.96) ≤3 days, decreasing to 0.89 (95% CI, 0.84–0.93) and 0.88 (95% CI, 0.83–0.92) at ≤7 and ≤10 days, respectively, but was found to be as low as 0.36 (95% CI, 0.21–0.55) 10 days after onset of symptoms, when viral replication has likely terminated [23]. Important evidence has also been provided that subjects testing negative with a SARS-CoV-2 AG-RDT 6 days after symptoms onset are extremely unlikely to have an associated positive viral culture [24].

Finally, with respect to predictive symptoms of SARS-CoV-2 Ag-RDT positivity, an interesting study conducted during high prevalence of the Omicron linages [25] reported that none of the most common manifestations of COVID-19 were capable to anticipate test positivity.

Vaccination status

The vaccination has also been reported as a possible determinant of SARS-CoV-2 Ag-RDTs diagnostic sensitivity. More specifically, a recent study published by Bollinger and colleagues underpinned that the diagnostic sensitivity of these tests may be nearly 14% lower in vaccinated subjects (i.e., 0.39; 95% CI, 0.31–0.48) compared to those unvaccinated (i.e., 0.53; 95% CI, 0.44–0.62) [26], which is probably due to the lower viral load that typically accompanies COVID-19 vaccine breakthrough infections [27]. In a parallel investigation, Chu et al. also found that the cumulative diagnostic sensitivity of a randomly administered SARS-CoV-2 Ag-RDTs decrease by over 20% in vaccinated subjects, from 0.53 (95% CI, 0.51–0.56) in unvaccinated patients to 0.30 (95% CI, 0.24–0.36) in those who received one or more doses of COVID-19 vaccines [28]. Moreover, it may be proffered that an early inflammatory response to the virus in vaccinated (or previously infected) persons due to immunologic memory may result in clinical symptoms prior to establishment of a sufficient viral load, thus leading to false negative Ag-RDT results if the test is performed too early after symptoms onset.

Self- vs. healthcare-collected sample

As previously mentioned, one of the major advantages of Ag-RDTs, irrespective of their diagnostic target (thus encompassing all types of Ag-RDTs used for diagnosing other infectious diseases, defining pregnancy status, identifying drug of abuse consumption and so forth), is the possibility of their usage outside the laboratory environment, directly by patient at the site of collection (e.g., at the bedside, but also in pharmacies, schools, before accessing healthcare facilities or participating to crowded events or mass gatherings, etc.). Nonetheless, self-collection of respiratory specimens (saliva may be an exception) exposes these tests to an inherently enhanced risk of lower accuracy compared to healthcare staff collection. However, a systematic literature review and meta-analysis carried out by Mistry et al. showed that the use of specimens collected by trained staff does not dramatically impair the accuracy of testing in comparison to self-collected specimens, generating almost overlapping diagnostic sensitivities (i.e., 0.79 vs. 0.81; odds ratio (OR) of self-collection, 0.84 and 95% CI, 0.58–1.23; p=0.340) [29]. Another interesting study, published by Frediani et al. provided similar findings, demonstrating reliable usability with either self- or caregiver-administration, across different age groups [30]. Notably, a modest discrepancy in favour of caregiver-collected respiratory samples (i.e., around 10% higher sensitivity) could only be seen in specimens with cycle threshold (Ct) values between 20 and 25. Indeed, accurate education to collect the sample, perform the test and interpret the results is one of the major determinants of SARS-CoV-2 Ag-RDTs accuracy, which will be discussed in a following section of this article.

Use of oral or nasal virucidal agents

There are several substances which may temporarily act as virucidal, thus reducing to various extent the oral and/or nasal viral load. Several studies have been published on this matter, recently reviewed by us [31]. In summary, the list of potential interfering substances (i.e., those which may be effective to significantly lower the viral load) include povidone-iodine, phthalocyanine, cetylpyridinium chloride, octenidine dihydrochloride, chlorhexidine, hexitidine, hydrogen peroxide, delmopinol hydrochloride, hypochlorous acid, β-Cyclodextrin, essential oils and quaternary ammonium. Although the use of these agents would not directly impair the accuracy of SARS-CoV-2 Ag-RDTs using anterior nasal samples, since the swab would not enter in direct contact with the virucidal substance in the oral cavity, it is noteworthy that collecting nasopharyngeal or combining nasal and oropharyngeal collection after recent usage (i.e., between minutes to few hours) of these substances may impair test sensitivity. Besides their oral usage, some of these substances could also be included in the formulation of nasal sprays, where they can exert a similar antiviral activity. The results of a recent study, for example, showed that nasal sprays containing sodium hypochlorite or essential oils were effective to lower the viral load between 2–3 orders of magnitude [32]. A significant impact on SARS-CoV-2 testing accuracy has also been noted with the use of carrageenan-containing over-the-counter nasal and oral sprays, since some of these preparations may have a significant antiviral activity [33, 34]. It is not readily clear to what extent these compounds may impair test sensitivity, though their impact on reducing the nasopharyngeal viral load must be considered. A reduction of the viral load has also been observed in a pilot trial with chewing gum containing natural antiseptic agents [35]. Importantly, a recent study has also revealed that regular nasopharyngeal wash with normal saline, performed at 4 h interval during a period of 16 h, was also effective to significantly decrease the viral load, by around by 9% in 24 h [36].

The impact of eating certain types of food before undergoing SARS-CoV-2 Ag-RDTs testing remains controversial. A recent study showed that some aliments (especially hot dogs, potato chips, French fries, ice creams), assumed before testing may impair molecular detection of the virus in saliva [37], though no clear evidence has been published to-date on antigen testing to the best knowledge of the authors. Nonetheless, avoiding food intake immediately before sampling (i.e., for at least 30 min) seems advisable for preventing test inaccuracy. Interestingly, Velavan et al. reported that some soft drinks, energy drinks and even alcoholic beverages such as vodka, brandy and whiskey may cause false positive test results with certain SARS-CoV-2 Ag-RDTs, likely attributed to alteration of pH conditions, a circumstance that could be prevented by adequately mixing the beverages with sample buffer [38].

Type of specimen

The issue concerning the dependence of performance of SARS-CoV-2 Ag-RDTs on the type of specimen has been broadly analyzed in the current scientific literature. On the premise that the preferred sample is that recommended by the manufacturer in its usage instructions (as the test has been presumably validated on that sample matrix and the analytical and clinical performance on other materials are uncertain), a series of evidence-based considerations can be proffered.

The most important evidence comes from the most recent update published by the Cochrane COVID-19 Diagnostic Test Accuracy Group [13]. Based on this analysis of validated scientific literature, the nasal sample yields the most superior sensitivity with SARS-CoV-2 Ag-RDTs (i.e., 0.77; 95% CI, 0.70–0.82), followed by the nasopharyngeal swab (i.e., 0.69; 95% CI, 0.65–0.72) and a combination of nasal and oropharyngeal swabbing (i.e., 0.69, 95% CI, 0.55–0.80). The diagnostic sensitivity of oropharyngeal swab is only modest (i.e., 0.57; 95% CI, 0.27–0.83), whilst that of saliva is dramatically poor (i.e., 0.20; 95% CI, 0.07–0.45). These findings have been confirmed by other concomitant or even more recent meta-analyses [23, 39] (i.e., pooled sensitivities of around 0.50 or lower), confirming that the use of saliva is highly unreliable for this type of portable tests. Nonetheless, due to the preferential localization in the upper respiratory tract of the SARS-CoV-2 Omicron lineages, whose infection is hence associated with a larger predominance of throat symptoms [40], recent evidence emerged during the Omicron waves then suggests that combining nasal collection for SARS-CoV-2 Ag-RDTs (as typically recommended by most manufacturers) with oropharyngeal swabbing may be effective to enhance test accuracy. For example, Schuit et al. recently investigated the diagnostic sensitivity of some SARS-CoV-2 Ag-RDTs in over 6,000 people aged 16 years or older, tested for suspected infection [16], and found that the diagnostic sensitivity of the manual test kits increased from 0.70 using only anterior nasal samples up to 0.77–0.83 combining anterior nasal and oropharyngeal sampling. No difference in diagnostic sensitivity seems to exist between collecting anterior or mid-turbinate nasal swabs [13].

Device instructions

As with any other human activity, the accuracy of performance depends considerably on the completeness, clarity and readability of instructions given to the end-user. SARS-CoV-2 Ag-RDTs are no exception to this rule of thumb, especially in case of self-testing [41]. Interestingly, the results of a randomized clinical trial evidenced that the home usage of SARS-CoV-2 Ag-RDTs according to manufacturers’ instructions was associated with a substantial deviation from the Centers for Disease Control and Prevention (CDC) indications for quarantine and isolations, causing unnecessary home segregation or unwanted risks of contagion [42]. The important drawback of inaccurate/incomplete instructions provided by the manufacturers of SARS-CoV-2 Ag-RDTs has been highlighted in an interesting survey published by Özcürümez et al. [43], who explored the technical and analytical information provided by as many as 77 different test kits. The results that emerged are rather disappointing, in that important data were lacking in a considerable number of these tests. The most essential information that was missing included the detection limit (i.e., the threshold of viral load after which the Ag-RDT would turn positive) and the detection rate in subjects with high viral load (both absent in over 80% of the package insert of tests), the high-dose effect threshold (missing in over 60% of test instructions), repeatability and reproducibility (unreported by nearly 70% of manufacturers), as well as the specific SARS-CoV-2 antigen(s) detected (lacking in nearly one third of all assays). Ameliorable usability of these devices has been highlighted in other investigations, such as that carried out by Krüger and colleagues [44], who found that some operations such as transferring the buffer within the diluent tube (when requested), the swab extraction procedure or transferring an appropriate volume of liquid into the sample wall were the most critical steps in some test kits, thus reducing sensibly users’ satisfaction.

The misleading interpretation of provided instructions is another important aspect that may contribute to impair the accuracy of these rapid tests, wherein the whole procedure is sometimes unclear, confusing, not clearly understandable/intelligible or, in some cases, even written with microscopic fonts that can be hardly read, especially by older persons. Evidence suggests that a non-negligible percentage of users (e.g., up top 15%) may need some form of help for performing the test [45], thus underpinning further the risk of inappropriate self-test execution. In general, the multiple steps needed for self-performing the test (basically summarized in the introduction of this article and reproduced in Figure 1) are relatively simple provided that the user has been appropriately trained and the instructions are carefully followed. Nonetheless, some doubts may haunt even skilled healthcare professionals. Clear instructions should also be present concerning the optimal storage conditions and the expiration date after which the kit must be absolutely discarded. Caution shall also be paid to avoid mixing swabs, tubes or devices when multiple tests are performed at the same time on multiple patients.

Notably, an interesting study published by Papenburg and colleagues showed that implementation of locally modified reference guides, rather than only relying on the information given in the instruction manual of the test, may be a practical way for improving the performance of SARS-CoV-2 Ag-RDT self-testing [46]. More specifically, the authors demonstrated that the diagnostic sensitivity of a SARS-CoV-2 RDT could be improved from around 0.50 to nearly 0.90 by administering a quick personalized reference guide for testing.

Sample storage

There is no doubt that the sample storage (and related stability) remains a major preanalytical determinant of testing quality, throughout all types of in vitro diagnostic testing [47]. In the circumstance of Ag-RDTs, however, sample storage has a relatively lower importance compared to other variables, since the test is intended to be completed immediately (i.e., seconds to few minutes) after the respiratory sample has been collected. Although the storage of test kits under appropriate conditions of temperature and humidity may be critical as for any laboratory reagent (the presence of a “control” band would provide, however, an internal quality control to testify that the kit is appropriately working), there is no clear reason to store the swab or the vial for long periods of time, since this would contradict the intrinsic reasons for performing this type of immediate testing (i.e., self-testing, rapidity). Nonetheless, it may be worthwhile mentioning here that the use of specific preservation media is effective to maintain stable the SARS-CoV-2 antigen(s) concentration in nasopharyngeal samples for several hours (i.e., up to 72 h) regardless of the storage temperature [48]. A recent study also showed that nasopharyngeal samples stored at 4 °C for up to one week or frozen for up to 7 months at −20 °C provide comparable results to fresh samples using a commercial SARS-CoV-2 Ag-RDT [49].

A circadian rhythm has also been described for saliva and nasopharyngeal viral load, which may hence influence the outcome of SARS-CoV-2 testing. In a recent study published by Viloria Winnett et al. [50], for example, the authors found that saliva and respiratory samples collected early in the morning after waking may be associated with detection of higher SARS-CoV-2 viral loads compared to similar samples taken later during the day. Such higher viral load in the morning would then enhance the likelihood of diagnosing SARS-CoV-2 infection, especially when tests with lower analytical sensitivity such as Ag-RDTs are used.