key: cord-0772862-y6ijg536
authors: Leixner, Georg; Voill-Glaninger, Astrid; Bonner, Elisabeth; Kreil, Anna; Zadnikar, René; Viveiros, André
title: Evaluation of the AMP SARS-CoV-2 Rapid Antigen Test in a Hospital Setting
date: 2021-06-01
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.05.063
sha: f1ab44865b52fefbab2e5058d4ec6c5aaa1c8009
doc_id: 772862
cord_uid: y6ijg536

OBJECTIVES: Quick and inexpensive SARS-CoV-2 screening and frontline testing are of growing demand. The aim of our study was to evaluate the performance of the immunochromatographic AMP rapid antigen test (AMP RAT) in comparison with the gold-standard real-time reverse transcription PCR (rRT-PCR) in a hospital cohort. METHODS: A total of 392 patients, who presented consecutively with COVID-19 symptoms at our emergency department, were included in this retrospective study. Two swabs were collected per patient: a nasopharyngeal for the RAT and a combined naso- and oropharyngeal for the rRT-PCR. A positive rRT-PCR (defined as cycle threshold (Ct) < 40) was found in 94 (24%) patients. RESULTS: In our cohort with a median patient age of 70, overall sensitivity and specificity of the AMP RAT was 69.1% (58.8 – 78.3, 95% CI) and 99.7% (98.1 – 100.0, 95% CI), respectively. In patients with a Ct value < 25 and < 30, higher sensitivities of 100.0% (89.4 – 100.0, 95% CI) and 91.8% (81.9 – 97.3%, 95% CI) could be observed. CONCLUSIONS: The AMP RAT showed a high sensitivity in patients with a Ct value < 25 and < 30 and might be useful for frontline testing, whenever rRT-PCR is not rapidly feasible.

• Sensitivity: 91.8% 95% CI) Ct < 25:

• Sensitivity: 100.0% 95% CI) Overall:

• Sensitivity: 69.2% 95% CI) • Specificity: 99.7% (98.1 -100.0, 95% CI)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a broad clinical spectrum known as coronavirus disease 2019 and was first identified in December 2019 in Wuhan, China (Zhu et al. 2020; Zhou et al. 2020) .

Within a short period of time a world-wide spread lead to the current pandemic that will presumably remain the leading infectious disease topic in 2021 (WHO 2020a) .

Detection of the virus through nucleic acid amplification tests such as real-time reverse transcription PCR (rRT-PCR) is the gold standard for the diagnosis of COVID-19 (WHO 2020b; CDC 2020c) . The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend nasopharyngeal and/or oropharyngeal swabs as the most sensitive specimen types for rRT-PCR testing (CDC 2020b; WHO 2020b) . rRT-PCR is a cost-and laborintensive technology which requires trained personal (Corman et al. 2020 ).

J o u r n a l P r e -p r o o f Furthermore, specimen transport and the inherent logistics are often time consuming (CDC 2020b) . Although point-of-care testing (POCT) platforms allow a faster and easier preparation for rRT-PCR, they can still be expensive and its output is limited.

POCT is therefore not ideal for screening and disease outbreaks, especially in lowincome countries (Pai et al. 2012) .

To dampen and control the spread of the virus, a rapid, cheap, reliable and easy-tohandle identification test is needed for swift isolation or surveillance of patients as well as wide population screening. Rapid antigen tests (RAT) meet all these criteria and play a central role in the context of acute viral infections (Lai et al. 2020; Clerc and Greub 2010) . Hence, high sensitivity and specificity are crucial (CDC 2020a). In this study, we set out to evaluate the diagnostic performance of the commercially available AMP SARS-CoV-2 Rapid Antigen Test (AMP Diagnostics 2020) in comparison with rRT-PCR.

J o u r n a l P r e -p r o o f

A total of 392 patients with COVID-19 symptoms presented consecutively to the emergency department of the Klinik Landstrasse from November 2 nd 2020 through December 29 th 2020 and were included in this retrospective study. The Klinik

Landstrasse is a tertiary hospital and belongs to the Vienna Healthcare Group in Vienna, Austria.

For enrollment in the study and according to the infectious disease policy of our hospital, every incoming patient with COVID-19 symptoms had to be tested for SARS-CoV-2 by rRT-PCR and simultaneous RAT to narrow the time until diagnosis.

Nasopharyngeal swabs for RAT were collected at the same time as combined nasoand oropharyngeal swabs for rRT-PCR. While RAT was immediately performed and read after 15 minutes at the emergency department, rRT-PCR was carried out subsequently in our laboratory. Therefore, rRT-PCR results were unknown at the time of RAT performance and reading.

COVID-19 symptoms were grouped according to the WHO classification (WHO 2021) in common (fever, dry cough, fatigue), less common (sore throat, diarrhea, headache or other aches/pains, conjunctivitis, anosmia or ageusia, skin rash, discoloration of fingers or toes) and severe (dyspnea, chest pain, focal neurological deficit).

The AMP Rapid Test SARS-CoV-2 Ag (AMP Diagnostics, AMEDA Labordiagnostik GmbH, Graz, Austria), henceforth AMP RAT, is a rapid immunochromatographic test for qualitative detection of the SARS-CoV-2 nucleocapsid protein antigen in J o u r n a l P r e -p r o o f nasopharyngeal swabs (AMP Diagnostics 2020). The AMP RAT was performed at the emergency unit immediately after collection of nasopharyngeal swabs by special trained medical staff. Specimen collection and analysis were performed at room temperature. For each RAT, and according to manufacturer's instructions, the swab was inserted in the extraction buffer tube and rotated at least 6 times while gently pressing the flocked head of the swab against the inner wall of the tube. After one minute of incubation, approximately 100 µl (4 drops) of the extraction solution was dropped into the sample well of the cassette which then migrates by capillary effect along the membrane. The SARS-CoV-2 nucleocapsid antigen binds to monoclonal antibody conjugated with colloid-gold particles. It is then captured by secondary monoclonal antibodies and immobilized in the test region and a colored line appears.

A colored line has to appear in the correspondent internal control (IC) region confirming sufficient sample volume and correct test procedure ( Figure 1 ). The test results can be read after 15 but no later than 20 minutes.

For rRT-PCR, combined naso-and oropharyngeal swabs were collected and inoculated in a sterile 2 ml 0.9% NaCl solution produced by the hospital pharmacy and then sent to our laboratory. All samples were analyzed within a time period less than 6 hours on one of five different platforms available in our laboratory (Table S1 ).

All rRT-PCR with a cycle threshold (Ct) value < 40 were considered positive. When two Ct values of different target genes were available, a mean Ct value was calculated. In the case of SarbecoV E-gene EAV (TIB Molbiol Syntheselabor GmbH) and cobas ® Liat ® (Roche Diagnostics), only one Ct value was used. The SarbecoV E-gene EAV reagent targets a single gene (E) and the cobas ® Liat ® calculates one amplification curve by using the same probe for both targeted genes (ORF1a/b and J o u r n a l P r e -p r o o f E). Cobas ® Liat ® only shows the coordinates of the amplification curve and does not give definite Ct values. In this case, Ct values were read and given as integers by two laboratory physicians.

As rRT-PCR is the diagnostic gold standard for SARS-CoV-2 detection, positive and negative samples were considered as true positive and true negative, respectively. presented with severe symptoms and 33% were in need of oxygen support (Table 1) .

The rRT-PCR results revealed 94 positive samples and 298 negative samples. The median Ct value was 27.6 (range: 14.1 -39.9). Ct values did not significantly differ between the different rRT-PCR devices ( Figure S1 ). When rRT-PCR positive and negative patients were compared, no significant differences were observed in terms of age, sex, symptoms and need for oxygen therapy. Time of presentation since symptom onset ranged from 0 to 60 days in COVID-19 patients and from 0 to 28 days in rRT-PCR negative patients, but this difference did not reach significant difference ( Table 1) .

The IC was positive in all 392 performed RAT and so no test had to be repeated. The AMP RAT showed an overall sensitivity of 69.1% and specificity of 99.7%, with a single false positive ( Table 2) . As expected, binary logistic regression could identify the mean Ct value as an independent predictor for a positive RAT in the COVID-19 (rRT-PCR positive) group. In contrast, time since symptom onset did not predict for RAT positivity (Table 3) .

For further analysis, COVID-19 patients were grouped according to a mean Ct value < 30 and < 25, in which case RAT sensitivity increased to 91.8% and 100.0%, respectively (Table 2) .

Discussion Different SARS-CoV-2 RAT with distinct handlings and test performances are available (Dinnes et al. 2021) . Here, we set out to compare the AMP RAT with rRT-PCR in a hospital cohort. This is the first AMP RAT study to deliver real-life data.

Many studies evaluating RAT have been published using transport media (e.g., universal transport medium, viral transport medium and phosphate buffered saline) as a proxy for direct testing after sample collection (Kruttgen et al. 2021; Mak et al. 2020; Porte et al. 2020) . However, most of the available tests, including the AMP RAT, are only approved to be performed directly after swabbing. A strength of our study relies on the fact that RAT were performed strictly according to the manufacturer's instructions in a point-of-care setting.

In direct comparison with the rRT-PCR, the AMP RAT showed a sensitivity of 100.0%

for Ct values < 25 and 91.8% for Ct values < 30. As expected, and in accordance with previous studies, sensitivity dropped progressively with higher Ct values (Kruttgen et al. 2021; Mak et al. 2020; Porte et al. 2020; Scohy et al. 2020 ).

One possible study limitation is that Ct values had to be manually read from the cobas ® Liat ® rRT-PCR system as in this case only indexed amplification curves but no given Ct values were produced. To minimize observational bias, Ct values were read by two physicians as integers. We consider that the data presented here would not change significantly with automatically given Ct values. Another inherent limitation to our study is the retrospective character of data collection.

The AMP RAT product information discloses an overall sensitivity of 97.3%, which differs considerably from the sensitivity of 69.1% reported here. This could be due to different patient selection as, in our cohort, a wide range of Ct values, including 33 J o u r n a l P r e -p r o o f samples with a Ct value ≥ 30, could be observed. Another relevant issue is that the viral load of the nasopharyngeal swab does not necessarily reflect the viral load of the upper respiratory tract due to an inherent preanalytical variance concerning the quality of the swabbing method (e.g., nasal polyps, anatomical differences, patient discomfort). Further, the SARS-CoV-2 RAT landscape has been recently analyzed in a vast Cochrane systematic review and revealed widely divergent overall sensitivities (range: 0 -96%) (Dinnes et al. 2021 ).

In the binary logistic regression analysis, RAT positivity was not associated with the time between symptom onset and SARS-CoV-2 testing, most probably because median time after symptom onset in our study was very low (two days in COVID-19 patients and one day in rRT-PCR negative patients).

From a user-friendliness point of view, the AMP RAT is comparable to other commercially available RAT that we tested at our laboratory. An additional incubation step of one minute in the extraction buffer tube is needed and makes testing more demanding. Nonetheless, in our experience, the AMP RAT still has a good deployability in an emergency department setting.

In conclusion, the AMP RAT showed a good test performance in patients with low Ct values and might be a useful tool for frontline testing, whenever quick rRT-PCR testing is not feasible. Tables and Figures   Table 1 . Demographic and clinical data of patients.

Data are given as n (%) or median (25 th -75 th percentiles) * Symptoms were grouped according to the WHO classification (WHO 2021) in common (fever, dry cough, fatigue), less common (sore throat, diarrhea, headache or other aches/pains, conjunctivitis, anosmia or ageusia, skin rash, discoloration of fingers or toes) and severe (dyspnea, chest pain, focal neurological deficit). These were counted separately, as a combination of symptoms from different groups is possible. (34) 232 (59) 106 (27) 34 (36) 50 (53) 21 (22) 100 (34) 182 (61) 85 (29) 0.55

Oxygen therapy, n (%) 129 (33) 

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