While nucleic acid amplification tests remain the gold‐standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections, immunological methods can also be used to detect viral antigens or specific antibodies.^1-9 Several rapid diagnostic tests employing antigen detection (Ag‐RDTs) are now commercially available. However, there remains limited evidence on the utility of Ag‐RDTs for coronavirus disease 2019 (COVID‐19) diagnosis and surveillance, with significant variability reported with respect to their diagnostic performance and a lack of external validation for many of the available tests.¹⁰ Thus, we initiated our study to evaluate an Ag‐RDT, the PCL COVID‐19 Ag rapid fluorescent immunoassay (FIA), for diagnosis of COVID‐19 in hospitalized patients with suspect SARS‐CoV‐2 infection. The results of this test were compared with qualitative and quantitative results of parallel reverse transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) examinations.

Patients hospitalized with suspected COVID‐19 between May and September 2020 at a primary care hospital in Siedlce, Poland, were enrolled in this prospective observational study. Patients were simultaneously sampled by nasopharyngeal swabs for antigen detection and RT‐PCR. Swabs were collected four times at 2 days intervals from all but one patient, that was examined only three times. Blood samples for serology testing were taken two times at a 6‐day interval, together with the first and last nasopharyngeal sample. The antigenic assessment was performed using PCL COVID‐19 Ag Rapid FIA (PCL) as a point of care test in accordance with the manufacturer's instructions. The molecular assessment was performed within a few hours after collection at the central COVID‐19 laboratory in Warsaw using commercial RT‐PCR kits (Liferiver, DiaplexQ, or Vitassay). Serology testing for anti‐SARS‐CoV‐2 immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA) was performed using ELISA (Euroimmun). This study was performed in accordance with the Declaration of Helsinki and under patient informed consent with anonymization of data.

A total of 42 patients (21 male and 21 female) with a median age of 57.7 years (range, 21–78) were included resulting in collection of 167 nasopharyngeal swabs. The median time between onset of symptoms and the first sample taken was 8.3 days (range, 2–16).

A summary of results over the course of the study are presented in Figure 1. Among the 167 nasopharyngeal swabs, 95 (56.9%) were positive, 26 (15.6%) were inconclusive, and 46 (27.5%) were negative for SARS‐CoV‐2 by RT‐PCR, while only 25 (15.0%) of swabs were positive by Ag‐RDT. The overall percent agreement between Ag‐RDT and RT‐PCR was 48.9%. Positive results of RT‐PCR were obtained in 36 (85.7%) patients, while 1 (2.4%) patient had inconclusive RT‐PCR results, 1 (2.4%) patient had negative results, and 4 (9.5%) had mixed negative and inconclusive RT‐PCR results over their course of hospitalization. Ag‐RDT revealed antigen detection in 15 (35.7%) patients. In one case, Ag‐RDT was positive in a patient with a negative RT‐PCR results at all time points. Using RT‐PCR as reference standard, the specificity of the Ag‐RDT was 83.3%. Among the 36 RT‐PCR‐positive patients, the Ag‐RDT test was positive in only 14 cases, resulting in a sensitivity of 38.9%. Among the 15 Ag‐RDT positive patients, nine had SARS‐CoV‐2 antigen detected only in the first sample, two patients in two samples, and four cases in three consecutive samples. No patient had persistence of a positive Ag‐RDT result in all four samples obtained during the study. In patients with a positive Ag‐RDT, cycle threshold (CT) values for the Orf1ab gene in the RT‐PCR test ranged from 18.5 to 36.5 (mean 27.8). In all cases, Ag‐RDT positivity was observed no later than 10 days after symptom onset (Table S1). The results of serology testing revealed the presence anti‐SARS‐CoV‐2 antibodies in 41 out of 42 (97.6%) patients. Anti‐SARS‐CoV‐2 IgA (90.5%) and IgG (85.7%) were frequently observed, while anti‐SARS‐CoV‐2 IgM was detected in only 20 (47.6%) patients.

In our investigation, the Ag‐RDT PCL COVID‐19 Ag rapid FIA had a sensitivity of 38.9% and specificity 83.3%, with only 48.9% overall agreement between Ag‐RDT and RT‐PCR. The observed sensitivity of the PCL Ag‐RDT was significantly lower than that announced by the manufacturer, who reported a sensitivity of 100% (27/27) and a specificity of 97.8% (44/45). Nonetheless, the results of an Ag‐RDT depend on several factors, such as time from the symptom onset, the specimen viral content, and other preanalytical and analytical considerations.^{2, 4, 10, 11} In our study, the PCL COVID‐19 Ag rapid FIA had a sensitivity 92.9% for CT values under 25, which demonstrated that CT values were crucial to the results of Ag‐RDT. Thus, PCL COVID‐19 Ag Rapid FIA was more sensitive at high viral loads and therefore its utility may be limited to patients with a recent onset of symptoms, when viral load is very high.

Although robust and simple in performance, this Ag‐RDT presented insufficient capabilities to replace the RT‐PCR. Similar conclusions about different RDTs have been presented by others.^3-5 However, despite relatively low clinical sensitivity among Ag‐RDTs, they may be helpful at reducing the number of patients requiring RT‐PCR and the burden on laboratories, particularly at a time of rapidly increasing number of COVID‐19 cases worldwide. Nonetheless, the performance of this particular Ag‐RDT limits its applicability for both clinical and community testing. While viral loads in patients with presymptomatic or asymptomatic disease are lower as compared to symptomatic patients, thus leading to limited detection of SARS‐CoV‐2 by Ag‐RDTs in such individuals,¹⁰ this may be deemed acceptable, as such a low viral load seemingly correlates to the reported reduced transmission potential among asymptomatic or presymptomatic persons.¹² However, this assay failed to accurately detect SARS‐CoV‐2 in symptomatic hospitalized patients, raising substantial concerns over its utility. The low sensitivity could lead to missing a substantial number of symptomatic COVID‐19 cases with potentially high transmission potential or leading to delays in initiating self‐quarantine procedures and contract tracing while pending repeat testing. Further studies should evaluate this assay in a larger, more diverse cohorts.

In conclusion, we observed low sensitivity for the PCL COVID‐19 Ag rapid FIA in detecting active SARS‐CoV‐2 infection in hospitalized patients. Moreover, the overall agreement between Ag‐RDT and RT was poor. These results highlight the need for careful evaluation and external validation of novel Ag‐RDT assays before implementation in clinical practice or in community surveillance.

尽管核酸扩增检测仍然是诊断严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）感染的金标准，但免疫学方法也可用于检测病毒抗原或特异性抗体。^1-9目前已有几种采用抗原检测（Ag-RDT）的快速诊断测试方法在市场上出售。但是，关于Ag-RDT在2019年冠状病毒疾病（COVID-19）诊断和监测中的效用的证据仍然有限，据报道其诊断性能存在明显差异，并且许多可用测试均缺乏外部验证。¹⁰因此，我们启动了研究以评估可疑SARS-CoV-2感染住院患者的PCL COVID-19 Ag快速荧光免疫分析法（FIA）Ag-RDT。将该测试的结果与平行逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检查的定性和定量结果进行了比较。

这项前瞻性观察研究纳入了2020年5月至2020年9月间在波兰Siedlce的初级保健医院中因怀疑COVID-19住院的患者。同时通过鼻咽拭子采样对患者进行抗原检测和RT-PCR。除一名患者外，每隔2天从所有患者中收集棉签4次，仅检查3次。每隔6天采集两次用于血清学检测的血液样本，以及第一个和最后一个鼻咽样本。抗原评估是根据制造商的说明，使用PCL COVID-19 Ag快速FIA（PCL）作为护理点测试进行的。使用商业RT-PCR试剂盒（Liferiver，DiaplexQ，或Vitassay）。使用ELISA（Euroimmun）对抗SARS-CoV-2免疫球蛋白M（IgM），免疫球蛋白G（IgG）和免疫球蛋白A（IgA）进行了血清学测试。这项研究是根据赫尔辛基宣言进行的，并在患者知情同意下进行了数据匿名处理。

总共包括42例患者（男性21例，女性21例），中位年龄为57.7岁（范围21-78岁），收集了167例鼻咽拭子。从症状发作到第一次取样之间的中位时间为8.3天（范围2-16）。

研究过程中的结果总结如图1所示。SARS-CoV的167例鼻咽拭子中，有95例（56.9％）呈阳性，有26例（15.6％）未定，而46例（27.5％）阴性。 RT‐PCR检测结果为‐2，而Ag‐RDT检测结果只有25（15.0％）拭子呈阳性。Ag‐RDT和RT‐PCR之间的总体一致性百分比为48.9％。在36（85.7％）例患者中获得了RT-PCR阳性结果，而1例（2.4％）的患者没有RT-PCR结果，1例（2.4％）的患者阴性，4例（9.5％）的阴性和阴性结果混合在住院期间，RT-PCR结果尚无定论。Ag‐RDT揭示了15（35.7％）位患者的抗原检测。在一种情况下，在所有时间点，RT-PCR结果均为阴性的患者中，Ag-RDT阳性。使用RT‐PCR作为参考标准，Ag‐RDT的特异性为83.3％。在这36例RT-PCR阳性患者中，只有14例Ag-RDT检测呈阳性，敏感性为38.9％。在15例Ag-RDT阳性患者中，只有9例仅在第一份样本中检测到SARS-CoV-2抗原，两例在两个样本中检测到，四例在连续三个样本中检测到。在研究过程中获得的所有四个样本中，没有患者持续出现Ag-RDT阳性结果。对于Ag-RDT阳性的患者，RT-PCR测试中Orf1ab基因的循环阈值（CT）值在18.5至36.5之间（平均27.8）。在所有情况下，在症状发作后至少10天观察到Ag-RDT阳性（表S1）。血清学测试的结果表明，在42名（97.6％）患者中有41名存在抗SARS-CoV-2抗体。经常观察到抗SARS-CoV-2 IgA（90.5％）和IgG（85.7％），

在我们的研究中，Ag-RDT PCL COVID-19 Ag快速FIA的灵敏度为38.9％，特异性为83.3％，而Ag-RDT和RT-PCR的总体一致性仅为48.9％。观察到的PCL Ag-RDT的敏感性明显低于制造商宣布的敏感性，后者报告的敏感性为100％（27/27），特异性为97.8％（44/45）。尽管如此，Ag-RDT的结果取决于几个因素，例如症状发作的时间，样本病毒含量以及其他分析前和分析方面的考虑。^{2、4、10、11}在我们的研究中，PCL COVID-19 Ag快速FIA对25以下的CT值具有92.9％的灵敏度，这表明CT值对于Ag-RDT的结果至关重要。因此，PCL COVID-19 Ag快速FIA在高病毒载量下更敏感，因此，当病毒载量非常高时，其效用可能仅限于近期出现症状的患者。

尽管Ag-RDT性能稳定且性能简单，但其替代RT-PCR的能力不足。其他人也提出了关于不同RDT的类似结论。^3-5然而，尽管Ag-RDT之间的临床敏感性相对较低，但它们可能有助于减少需要RT-PCR的患者数量和实验室负担，特别是在全球COVID-19病例迅速增加的时候。但是，这种特定的Ag-RDT的性能限制了其在临床和社区测试中的适用性。尽管有症状或无症状患者的病毒载量比有症状的患者低，因此导致这些人中Ag-RDT对SARS-CoV-2的检测有限，¹⁰这可能被认为是可以接受的，因为如此低的病毒载量似乎与所报告的无症状或症状前者之间传播潜力的降低有关。¹²然而，该方法未能在有症状的住院患者中准确检测出SARS-CoV-2，这引起了人们对其效用的极大关注。低灵敏度可能会导致丢失大量可能具有高传播潜力的症状性COVID-19病例，或导致延迟等待重新进行测试时启动自我检疫程序和合同追踪。进一步的研究应在更大，更多样化的队列中评估该测定法。

总之，我们观察到PCL COVID-19 Ag快速FIA在住院患者中检测活动性SARS-CoV-2感染的敏感性较低。而且，Ag-RDT和RT之间的总体协议很差。这些结果表明，在临床实践或社区监测中实施之前，需要对新型Ag-RDT分析进行仔细评估和外部验证。