{"id":61218,"date":"2024-09-30T10:46:38","date_gmt":"2024-09-30T10:46:38","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=61218"},"modified":"2024-10-09T18:33:10","modified_gmt":"2024-10-09T18:33:10","slug":"the-establishment-of-the-spiking-method-to-evaluate-the-rapid-diagnostic-test-antigen-ag-rdt-product-for-covid-19-detection","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/the-establishment-of-the-spiking-method-to-evaluate-the-rapid-diagnostic-test-antigen-ag-rdt-product-for-covid-19-detection\/","title":{"rendered":"The Establishment of the Spiking Method to Evaluate the Rapid Diagnostic Test Antigen (Ag-RDT) Product for COVID-19 Detection"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

The rapid chromatographic immunoassay for the qualitative detection of specific antigens of SARS-CoV-2 can be performed by antigen-detecting rapid diagnostic tests (Ag-RDT)1, 2, 3<\/sup>. The number of Ag-RDTs on the market is recently increased in order to meet the demand for this product 4, 5, 6, 7<\/sup>. This situation forces the government to assess the safety, quality, and performance of distributed Ag-RDT products. Each country holds its own regulation for the implementation of Ag-RDT, including Indonesia. The Indonesian government has established testing rules to ensure the validity of Ag-RDT in the context of contact tracing, diagnosis, and Covid-19 screening8<\/sup>. The assay is carried out by two designated laboratories using a predetermined Standard Operational Procedure (SOP) as described in the decree of the Minister of Health of the Republic of Indonesia9<\/sup>. The testing result is required to obtain marketing authorization from the Minister of Health9, 10<\/sup>. The established protocol to validate the Ag-RDT kit is urgently needed due to the increasing demand from industries, societies, and laboratories during a prolonged Covid-19 pandemic.<\/p>\n\n\n\n

Based on the regulation in Indonesia, the samples used for Ag-RDT kit validation must be fresh, and should be prepared within 2×24 hours9<\/sup>. Freshly prepared samples are better for clinical testing in that there is no limitation due to storage or transportation. The tested samples included 30 positive samples with Cycle Threshold Value (Ct) values \u226425, 30 positive samples with Ct>25, and 30 negative samples. However, employing freshly prepared samples was sometimes difficult particularly when the Covid-19 case was declined. Therefore, other alternative protocols, which are highly validated should be established. In appropriate storage conditions at \u221280o<\/sup>C or \u221220o<\/sup>C, the stored biological samples can still be feasibly used. In previous studies, nasopharyngeal and oropharyngeal swabs stored in Viral Transport Media (VTM) or sterile saline can be feasibly stored at \u221270 \u00b0C for more than 12 days11<\/sup>. Gulec et al<\/em>. reported that swab samples for both positive and negative samples, can be stored and retained their quality at 4\u00b0C for up to 12 days12<\/sup>. A significant effect on the sample\u2019s Ct value was observed after 10 cycles of freeze-thawed13<\/sup>. Within a week of the time limit, not all laboratories were ready to supply the test samples, thus the comparison assay of the Ag-RDT validation was carried out using fresh samples and the spiking method using 2 weeks old samples. In this study, we reported the implementation of the spiking method using swab samples with a longer shelf life on two brands of Ag-RDT kits recommended by WHO, which were Panbio\u2122 Covid-19 Ag Rapid Test (Abbott) and Q Ag-Standard, RDT (SD Biosensor, Roche)14, 15<\/sup>.<\/p>\n\n\n\n

Methods<\/strong><\/p>\n\n\n\n

The assay was carried out by the \u201dSpiking Method\u201d which examined the flock swabs from the nasopharyngeal\/oropharyngeal specimens in VTM that were previously dipped into the Ag-RDT buffer kit. As a comparison, an RT-PCR assay was carried out on the same sample using QIAamp Viral RNA Kits (Qiagen) to isolate the viral RNA with N1 and N2 genes became the PCR-targeted genes. The reverse transcription and cDNA amplification were carried out using SuperScript\u2122 III Platinum\u2122 One-Step qRT-PCR Kit (Invitrogen). The Ag-RDT assay was carried out on 2 brands, namely Panbio\u2122 Covid-19 Ag Rapid Test (Abbott) and Standard Q Ag-RDT (SD Biosensor, Roche) using the spiking technique on specimens that were confirmed positive with Ct\u226425 and Ct>25, as well as negative specimens. The assay examined 2 different sample sets prepared from different time courses, including 2-week sand <48 hours samples<\/p>\n\n\n\n

RT-PCR<\/strong><\/p>\n\n\n\n

Samples were extracted using QIAamp Viral RNA Kits, which were then analyzed by RT-PCR using SuperScript\u2122 III Platinum\u2122 One-Step qRT-PCR Kit and Agilent AriaMx Real-Time PCR system.<\/p>\n\n\n\n

Sample Collection<\/strong><\/p>\n\n\n\n

The nasopharyngeal\/oropharyngeal specimens in VTM were obtained through the C.29 laboratory (National Reference Laboratory, Covid-19 Testing No. 29), Universitas Padjadjaran-Indonesia. Samples were taken from two different time courses; 90 samples of BBT (2 weeks) were taken on February 25-28, 2021, and 66 fresh samples (<48 hours) were taken on March 24, 2021. The old samples were obtained from several hospitals in Bandung, Indonesia, while the fresh samples were obtained from regular patients in our laboratory. <\/p>\n\n\n\n

Spiking Method Assay<\/strong><\/p>\n\n\n\n

The sterile flocked swab from each of the tested Ag-RDT kits was dipped and rotated to make sure all sides of the tip were coated. Then, the swab tip was taken out from the sample and swirled in the buffer fluid. The next step was dropping the spiked sample on the test device following each kit\u2019s reference instructions. The result can be obtained within 15 minutes.  <\/p>\n\n\n\n

Sensitivity and Specificity Analysis<\/strong><\/p>\n\n\n\n

The sensitivity percentage was calculated by the number of specimens identified as positive by the Ag-RDT assay divided by the number of specimens identified as positive by the RT-PCR reference assay. The specificity percentage was calculated by the number of specimens identified as negative by the Ag-RDT test divided by the number of specimens identified as negative by the RT-PCR reference assay.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

Initially, the 2-week-old sample was validated using two brands of Ag-RDT kits, namely Panbio\u2122<\/em> and SD Biosensor. The validation assay was carried out by the \u201cSpiking Method\u201d, which resulted in both high sensitivity and specificity values from both kit brands. According to the standard of the sensitivity and specificity values set by WHO, the results shown in Table 1<\/strong> have passed the standard qualifications. It has been proved that the dilution which performed at the validation assay using the \u201cSpiking Method\u201d did not substantially reduce the quality of the antigen testing.<\/p>\n\n\n\n

Based on the sensitivity values obtained\nat CT<25 and CT>25, the application of the antigen test is more accurate when\nemployed in\nsymptomatic patients with CT<25, while for CT>25 the sensitivity value did not successfully\nmeet the\nWHO standard. Previous studies suggested that Ag-RDT was not accurate\nin the samples with Ct value of over 25 as it contained low viral loads16,\n17<\/sup>. A high sensitivity value is influenced\nby a good sample\nstorage condition so that it retained the sample quality. There were\nno false positives observed from all the negative samples. However, there were\nsome false negative results were obtained in both Ag-RDT tests. Overall, the\nsensitivity and specificity values for both kit brands still met the required\nWHO standard for the samples with CT\u226425.<\/p>\n\n\n\n

Table 1: Ag-RDT performance according to storage time of samples<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Characteristics<\/strong><\/p>\n<\/td>\n

\n

Fresh Samples<\/strong><\/p>\n

(<\/strong>n=66<\/strong>)<\/strong><\/p>\n<\/td>\n

\n

2 Weeks Old Samples<\/strong><\/p>\n

(n=90)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Panbio\u2122<\/strong><\/p>\n<\/td>\n

\n

Standard\u2122 Q<\/strong><\/p>\n<\/td>\n

\n

Panbio\u2122<\/strong><\/p>\n<\/td>\n

\n

Standard\u2122 Q<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Ct\u226425<\/p>\n<\/td>\n

\n

Ct>25<\/p>\n<\/td>\n

\n

Ct\u226425<\/p>\n<\/td>\n

\n

Ct>25<\/p>\n<\/td>\n

\n

Ct\u226425<\/p>\n<\/td>\n

\n

Ct>25<\/p>\n<\/td>\n

\n

Ct\u226425<\/p>\n<\/td>\n

\n

Ct>25<\/p>\n<\/td>\n<\/tr>\n

\n

Sensitivity<\/strong><\/p>\n<\/td>\n

\n

90%<\/p>\n<\/td>\n

\n

19%<\/p>\n<\/td>\n

\n

85%<\/p>\n<\/td>\n

\n

13%<\/p>\n<\/td>\n

\n

93%<\/p>\n<\/td>\n

\n

10%<\/p>\n<\/td>\n

\n

90%<\/p>\n<\/td>\n

\n

7%<\/p>\n<\/td>\n<\/tr>\n

\n

Specificity<\/strong><\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Furthermore, a comparison assay was conducted with samples aged <48 hours or fresh samples collected sometime after evaluating the 2-week-old sample. The total of collected samples was 66 samples which was considered as less than the 2-week old sample since collecting the fresh samples at the same time as the desired number of positive and negative samples was quite difficult, especially when the Covid-19 cases began to decline. All samples were treated the same way using the \u201cSpiking Method\u201d in both the Ag-RDT brands. As shown in Table 1<\/strong>, it can be observed that the sensitivity and specificity values were very high at CT\u226425 for both kit brands.<\/p>\n\n\n\n

There are many factors must be considered when selecting an appropriate\nmethod to improve the evaluation of new devices in the clinical laboratory. In\nprevious studies, spiking techniques have been reported to be successful used\nfor a variety of different spiked-samples18<\/sup>. Dong et al. reported\nspiking pathogens into whole blood and virus into plasma showed acceptable\nreproducibility and can be followed by assay developers who are targeting low\nprevalence pathogens19<\/sup>. Detection of Mycoplasma pneumoniae<\/em> in\nspiked respiratory samples also showed high sensitivity20<\/sup>. The\npresent study and other studies suggest that the spiking method is effective\nand applicable. The spiking technique can be an alternative method for Ag-RDT\nevaluation when the fresh samples are difficult to obtain if the samples are\nwell-preserved at 200<\/sup>C or \u2013800<\/sup>C with only one freeze-thaw\ncycle allowed.<\/p>\n\n\n\n

The spiking technique offered advantages, such as the ability to get rapid\nresult due to its ability to test several Ag-RDT brands at the same time, which\nalso required only a set of experiments and a low-cost system. However, this\ntechnique raises concerns, especially for the companies,  as the\nvalidation that is carried out may not represent the true quality of the\nproduct that is being\ntested. It might be due to the dilution of the viral RNA which decreased the concentration. In addition, the compatibility of the buffer utilized for storing the\nsample with the Ag-RDT product has been brought into question. It has been\nnoted that the transport medium may comprise guanidine salt, which possesses\nthe property of a protein denaturant21<\/sup>. This may lead to a decrease\nin the activity of the protein or the complete denaturation of the protein due\nto the strong interaction between guanidine and the catalytic residues of the\nprotein22<\/sup>. Atienzar et al. have reported that 6 of 19 Ag-RDT brands\nwere incompatible with Amies media and the sensitivity decreased up to 2 to 20\ntimes23<\/sup>. It emphasizes the importance of choosing the appropriate sample matrices and assays\nfor each specific use, particularly when employing Ag-RDT, as it can greatly\naffect the effectiveness of isolation and tracing measures.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The C.29 Laboratory of Universitas Padjajaran has conducted an assay on two Ag-RDT kits that specifically met the WHO criteria with \u226580% sensitivity and \u226597% specificity, namely Panbio\u2122 Covid-19 Ag Rapid Test (Abbott) brand and Q Ag-Standard, RDT (SD Biosensor, Roche) using the \u201cSpiking Method\u201d on the samples with Ct value \u226425, >25, and RT-PCR-confirmed negative samples. In these studies, Panbio\u2122 and SD Biosensor showed  93% sensitivity for the samples with CT value \u226425 and 90% sensitivity for the 2 weeks-old samples. The evaluation and validation of Ag-RDT using this spiking technique are deemed required as it can gain more benefits when testing several Ag-RDT brands at the same time, as it can utilize the same sample set for multiple validations.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

This study was supported by the Directorate for Research, Community Service, and Innovation of Universitas Padjadjaran (DRPMI-UNPAD).<\/p>\n\n\n\n

Conflict of Interest<\/strong> <\/p>\n\n\n\n

The author(s) do not have any conflict of interest <\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article<\/p>\n\n\n\n

Data Availability Statement<\/strong><\/p>\n\n\n\n

This statement does not apply to this article.<\/p>\n\n\n\n

Ethics Statement<\/strong><\/p>\n\n\n\n

This study utilizes biological material stored without identifiable links to patients, approved by the Research Ethics Committee of the Faculty of Medicine, Universitas Padjadjaran, with registration no. 0720121265. <\/p>\n\n\n\n

Informed Consent Statement<\/strong><\/p>\n\n\n\n

This study did not involve human participants, and therefore, informed consent was not required.<\/p>\n\n\n\n

Author\u2019s contribution<\/strong><\/p>\n\n\n\n

Conceptualization, HLW; methodology HLW, SE; software, AF, NRN, and AL; validation, HLW, SE; formal analysis, HLW and AF; investigation, AF, NRN, and AL; resources, LF and NF; data curation, AF, NRN, and AL; writing\u2014review and editing, AF, HLW, NAH, LF, SE, and NF; supervision, HLW, LF, NF and SE; project administration, AL. All authors have read and agreed to the published version of the manuscript.<\/p>\n\n\n\n

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