Evaluation of the Analytical Sensitivity of a Molecular Point-of-Care Assay as Compared to 3 Lateral Flow Assays for Group A Streptococcus

Abstract

Background

An accurate and timely diagnosis of pharyngitis caused by group A streptococcus (GAS) is essential for ensuring appropriate antibiotic therapy. Early detection through point-of-care (POC) testing is valuable in initiating effective treatment. This study aimed at evaluating the analytical sensitivity of the molecular ID NOW™ Strep A 2 POC test as compared to 3 POC lateral flow assays: BD Veritor™ Plus System, Sofia® Strep A+ Fluorescent Immunoassay, and Sekisui Diagnostic OSOM® Strep A test for GAS detection.

Methods

Two Streptococcus pyogenes bacterial American Type Culture Collection (ATCC) isolates and one clinical isolate from a throat swab sample were used to evaluate the limit of detection (LoD) of each assay. Ten-fold serial dilutions of the isolates starting from 10⁷ colony factor units (CFU)/mL were prepared, and each dilution was tested in triplicate for all 4 assays simultaneously. All tests were performed as per manufacturers’ instructions. The LoD was defined as the last dilution that yielded positive results for all 3 replicates. CFU per swab was also calculated.

Results

For the 3 isolates evaluated, LoDs of ID NOW Strep A 2 ranged from 3.125 × 10³ to 2.5 × 10⁴ CFU/mL; for Quidel Sofia the range was 1 × 10⁶ to 1 × 10⁷ CFU/mL; for BD Veritor the range was 1 × 10⁷ to 1.5 × 10⁷ CFU/mL; and for Sekisui OSOM the LoD was 1 × 10⁷ CFU/mL for all 3 isolates.

Conclusions

Compared to antigen-based lateral flow assays (LFAs), the molecular ID Now Strep A 2 assay demonstrated a lower LoD, which translates into higher sensitivity. In a clinical setting, this could enable detection of samples with a lower bacterial load that could be missed by low-sensitivity LFAs.

INTRODUCTION

Streptococcus pyogenes, also known as group A streptococcus (GAS), is the main cause of pharyngitis in children and adolescents (1). Each year, there are an estimated 11 million visits to ambulatory care centers for pharyngitis in children between 3 and 18 years of age (2). Approximately 15% to 30% of those children will be diagnosed with GAS pharyngitis (2, 3). Common symptoms presented during GAS infection are fever, sore throat, petechiae on the palate, and/or white spots on the throat or tonsils, with symptoms varying depending on the severity of the infection. An accurate and timely diagnosis of GAS is necessary to initiate appropriate antibiotic therapy. In addition, life-threatening complications such as acute rheumatic fever and suppurative complications (e.g., peritonsillar abscess, mastoiditis) are more likely to occur after an untreated infection (4). Diagnostic testing early in the disease course with point-of-care (POC) testing has also been shown to improve appropriate antibiotic prescribing within the infected population (5).

Currently, the Infectious Disease Society of America recommends confirmatory testing for all negative GAS lateral flow test results (6). Most often, a bacterial culture is the recommended reference test. However, culturing throat swabs is logistically difficult and therefore impractical in many settings around the world that also have a high incidence of streptococcal disease (7).

In addition to logistical challenges, the turnaround time from obtaining the throat culture to providing results is substantially greater than that of a rapid test. This delay, which is necessary to allow for culture growth, is one of the major disadvantages of throat cultures (8). The culture results also depend on the sample transport; if the sample is not transported in a timely manner at the correct temperature, the culture could be compromised, leading to a false-negative test result. Recovery rates at low temperatures (4°C) and low humidity are better than at higher temperatures (>25°C) (7). It is also known that recovery of beta-hemolytic streptococci from the throat is dependent on how well the collection is performed (9), because GAS is not evenly distributed in the pharynx (10).

The Abbott ID NOW™ Strep A 2 (Abbott Diagnostics), previously known as Alere i strep A, is a POC instrument-based molecular diagnostic test. The test utilizes nicking enzyme amplification reaction, a type of isothermal nucleic acid amplification technology, for the qualitative detection of GAS. Results from the device are provided in 6 min or less with a simplistic graphical user interface for convenience within a busy hospital or ambulatory environment. It is intended to be used to test throat swab specimens collected from patients symptomatic for pharyngitis (11).

Performance claims reported in some of the package inserts of various strep A lateral flow assays (LFAs) report sensitivities in the 90% range or greater, while publications more consistently report around 85% (12, 13).

The primary objective of this study was to compare the analytical sensitivity of the US FDA-cleared molecular ID NOW Strep A 2 test with 3 FDA-cleared GAS antigen LFAs [BD Veritor™ Plus System (chromatographic immunoassay; Becton Dickinson), Sofia® Strep A+ Fluorescent Immunoassay (Quidel Corporation), and Sekisui Diagnostic OSOM® Strep A test (color immunochromatographic assay; Sekisui Diagnostics) in the detection of 2 American Type Culture Collection (ATCC) strains of GAS and one clinical isolate of GAS obtained from a standard throat culture.

MATERIALS AND METHODS

Determination of Colony Forming Units/mL of Stock Cultures

Streptococcus pyogenes ATCC 12344, ATCC 19615, and one clinical Streptococcus pyogenes isolate obtained from throat culture sample were used in the study. All strains were subcultured on 5% blood agar plate (Becton Dickinson). Individual colonies were selected and suspended in PBS (Corning) to prepare main stock solutions. The colony forming units (CFU)/mL for each of these stock solutions was calculated by preparing five 10-fold dilutions for each strain. One hundred µL of each of these dilutions was plated onto two 5% blood agar plates and incubated for 18 to 24 h at 37°C for a colony count. CFU/mL of the stock cultures were calculated using the average number of colonies from the duplicate plates and the dilution factor. This CFU/mL count was used to calculate the amount of stock solution to take for limit of detection (LoD) determination.

LoD Determination

Using the stock culture solutions of each isolate, a bacterial suspension equivalent to 10⁷ CFU/mL was prepared in 2 mL of PBS. Serial dilutions were done to obtain various concentrations ranging from 10⁷ CFU/mL to 10³ CFU/mL (as indicated in Tables 1–3). Respective kit controls were used on each device on the days of testing to confirm the validity of the devices’ performance and reagents. Fifty µL from each dilution starting from 1 × 10⁷ CFU/mL was transferred to 3 separate round bottom tubes for each test. A swab was placed in the tube to absorb the aliquoted dilution and was used as a sample on the testing system. Testing of the swabs was performed as per manufacturers’ instructions for each test. All dilutions were tested in triplicates on each of the devices. The last dilution that was positive for all 3 replicates was considered the LoD. The average volume capacity for each swab (50 µL) was used to calculate the value of CFU per swab.

RESULTS

GAS ATCC 12344 exhibited positive test results for 7 serial dilutions (starting at 1 × 10⁷ CFU/mL) with ID NOW Strep A 2, demonstrating a final LoD of 6.25 × 10³ CFU/mL.

BD Veritor and Sekisui OSOM displayed positive results for only the first dilution, yielding a LoD of 1 × 10⁷ CFU/mL for both. Quidel Sofia demonstrated positive results for 2 serial dilutions and exhibited a final LoD of 1 × 10⁶ CFU/mL (Table 1).

GAS ATCC 19615 tested with the ID NOW Strep A 2 was positive for 8 dilutions (starting at 1 × 10⁷ CFU/mL), displaying a LoD of 3.125 × 10³ CFU/mL. BD Veritor initially tested at 1 × 10⁷ CFU/mL as the highest dilution was able to detect only 1 of the 3 replicates. Two additional dilutions at higher concentrations, 1.5 × 10⁷ CFU/mL and 3.7 × 10⁷ CFU/mL, were tested, and the LoD was found to be 1.5 × 10⁷ CFU/mL. Both Quidel Sofia and Sekisui OSOM displayed an LoD of 1 × 10⁷ CFU/mL (Table 2) for ATCC 19615. However, 2 of 3 replicates were detected positive with Quidel Sofia at 1 × 10⁶ CFU/mL (Table 2).

For the clinical GAS isolate obtained from a throat sample culture, the ID NOW Strep A 2 test displayed positive test results for all 3 replicates for the first 5 serial dilutions (starting at 1 × 10⁷ CFU/mL) indicating a LoD of 2.5 × 10⁴ CFU/mL, with 2 out of 3 replicates being detected at 1.25 × 10⁴, 6.25 × 10³, and 3.125 × 10³ CFU/mL. BD Veritor, Quidel Sofia, and Sekisui OSOM demonstrated positive results only at the first serial dilution, exhibiting a final LoD of 1 × 10⁷ CFU/mL for all 3 assays. The Quidel Sofia assay also detected 2 out of 3 replicates at 1 × 10⁶ CFU/mL (Table 3).

In comparing the analytical LoDs per swab of the 4 commercial assays, which were calculated using an average volume capacity of 50 µL as described in Methods, ID NOW Strep A 2 demonstrated the lowest LoDs per swab for all the 3 isolates tested. It was 312 (3.12 × 10²) CFUs for ATCC 12344 (5 × 10⁴ CFUs for Quidel Sofia, 5 × 10⁵ CFUs for BD Veritor and Sekisui OSOM). For ATCC 19615, the per swab LoD of ID NOW Strep A 2 was 156 (1.56 × 10²) CFUs (7.5 × 10⁵ CFUs for BD Veritor, 5 × 10⁵ CFUs for Quidel Sofia and Sekisui OSOM), and for the clinical isolate it was 1250 CFUs (1.25 × 10³) for ID NOW Strep A 2 (5 × 10⁵ CFUs for BD Veritor, Quidel Sofia, and Sekisui OSOM).

DISCUSSION

At the POC, LFAs have been widely utilized to rapidly diagnose, treat, and manage the spread of GAS. This is done using minimally trained staff in locations where laboratory services are not easily accessible (14). Increased analytical sensitivity and specificity allow for rapid POC testing to be more impactful for patient care.

In children, where GAS infection is common and presents with a variety of clinical symptoms, the pretest probability is high enough that a positive rapid swab result does not need to be confirmed by throat culture for diagnosis of GAS pharyngitis (15).

While clinical presentation can be a strong indicator of GAS infection, it cannot always reliably predict a true GAS case. Based on the Centor scoring system, the likelihood of a positive case of GAS consists of 4 variables—tonsillar exudates, swollen tender anterior cervical nodes, lack of a cough, and history of fever, but these factors alone cannot rule in or rule out GAS (3). One study showed that patients with all 4 variables had a 56% probability of a positive culture (post-test probability); 3 variables, 32%; 2 variables, 15%; 1 variable, 6.5%; and 0 variables, 2.5% (16).

Rapid antigen detection tests (RADTs) were developed and have been used since the 1980s to provide an immediate result for clinicians about the presence or absence of GAS (17). Compared with throat culture, RADTs offer diagnosis at the POC (within 5-10 min) (17). The use of these tests not only decreases the turnaround time to results for both patients and clinicians, but it also led to a considerable reduction in antibiotic prescriptions (18). In patients who are presenting with common GAS symptoms, RADTS generally exhibit adequate sensitivity and specificity to make appropriate Standard of Care (SOC) decisions (19). Similarly, molecular tests may also be used to provide faster turnaround time to results for patients at POC sites such as clinics. In addition, molecular tests are based on the chemistry of nucleic acid amplification, which further improves the analytical sensitivity of the test. This becomes particularly important in samples that have low bacterial loads, such as samples that were collected suboptimally, to ensure detection of GAS.

The current literature for the ID NOW Strep A 2 provides real-world evidence for its use in the accurate diagnosis of GAS infection within a hospital or POC setting (7, 11, 20, 21). One study performed on the ID NOW Strep A 2 found the test to be straightforward and simple to use by non-laboratory personnel with equivalent performance in both children and adults within various clinical settings (11). The sensitivity and specificity of the device are 98.5% and 93.4%, respectively. The ID NOW Strep A 2 LoD, defined as the concentration of GAS that produces a positive result approximately 95% of the time, was identified by evaluating different concentrations of GAS on the ID NOW Strep A 2 platform (7). In the current study, when compared to the 3 LFAs, the ID NOW Strep A 2 assay exhibited a lower LoD with 312 (3.12 × 10²), 156 (1.5 × 10²), and 1250 (1.25 × 10³) CFUs per swab for ATCC 12344, ATCC 19615, and the clinical isolate, respectively. The substantially lower LoD of this test can potentially expand access to testing without requiring an additional confirmatory test, while lateral flow tests still require culture confirmation. The Infectious Diseases Society of America guidelines state that due to the sensitivities of various RADTs being typically <90%, a negative RADT should be accompanied by a follow-up or back-up throat culture. The advantage of higher sensitivity molecular nucleic acid amplification technology-based assays is that a negative test does not always require bacterial culture confirmation (2).

It has been shown that performance data reported in product package inserts can sometimes differ when compared to real-world performance of assays. For example, LFA sensitivity differences may be due to a multitude of test factors that exist at POC, such as sample collection, which can differ substantially in a real-world setting vs controlled clinical trial settings. These discrepancies have led to guidelines requiring confirmatory culture testing for negative results utilizing RADTs.

A prospective study reviewed the difference between the sensitivity of LFAs mentioned in package inserts vs the sensitivity during typical clinical testing, where the clinical tests’ accuracy was much lower (positive predictive value: 11%–50%) than the sensitivity (87%–97.5%) and specificity (100%) mentioned for the assay (11). Real-world data refer to data collected from diversified areas of daily life that are outside the scope of highly controlled randomized control trials (22). For some LFA studies, sensitivities as low as 66% and 70% have been reported, respectively (23, 24). In a meta-analysis by Lean et al. in a pediatric population, the sensitivity of lateral flow immunochromatographic studies and optical immunoassays were similar at 85%, whereas the specificity of lateral flow test was slightly higher than optical immunoassays (25, 26). Results from the current study provide evidence and an explanation for how these variables, such as suboptimal sample collection and different bacterial loads in samples, can impact the results. The LoD of an assay impacts the results of an assay at POC. Tests with higher LoDs are more likely to not detect infected patients who may have lower bacterial loads, resulting in more false negatives (27). Molecular assays such as the ID NOW Strep A 2, as shown in this study, have a lower LoD and thus reduce the probability of false-negative results and additionally avoid the need for bacterial culture confirmation. Therefore, while the use of ID NOW Strep A 2 as compared to LFAs could have a similar turnaround time for a diagnosis of GAS, the probability of missing a positive patient result is lower. In addition, having an actionable negative result from the ID NOW Strep A 2 leads to proper treatment up front without the need for bacterial culture confirmation, which can take an additional 1 to 2 days for a final result.

In conclusion, the ID NOW Strep A 2 assay has a lower LoD when compared to the 3 LFAs tested in this study. Due to its higher sensitivity, it could potentially detect more positives that otherwise would have been reported as false negative. A revised comparison of the clinical sensitivity of all these assays may substantiate the findings of this study.

Nonstandard Abbreviations: GAS, group A streptococcus; POC, point-of-care; LFA, lateral flow assay; ATCC, American Type Culture Collection; CFU, colony forming units; LoD, limit of detection; RADT, rapid antigen detection test.

Author Contributions: The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.

Alamelu Chandrasekaran (Conceptualization-Equal, Data curation-Equal, Investigation-Equal, Methodology-Equal, Supervision-Equal, Validation-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Abdullah Kilic (Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Rinki Kumar (Data curation-Equal, Formal analysis-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Daniel Green (Conceptualization-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Fann Wu (Conceptualization-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), and Gregory Berry (Conceptualization-Equal, Formal analysis-Equal, Funding acquisition-Equal, Investigation-Equal, Project administration-Equal, Resources-Equal, Supervision-Equal, Writing—original draft-Equal, Writing—review & editing-Equal)

Authors’ Disclosures or Potential Conflicts of Interest:  Upon manuscript submission, all authors completed the author disclosure form.

Research Funding: Financial support and materials for testing were provided by Abbott Diagnostics.

Disclosures: D.A. Green has received consulting fees from Shionogi, BioFire, and Vibrant American and travel support from the American Society for Microbiology. G.J. Berry has provided educational talks and/or served on scientific panels and received honoraria from the following: Abbott, Biomerieux, Cepheid, Diasorin, Hologic, and Roche.

Role of Sponsor: The funding organization played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.

References