Study design and setting
A comparative study design was conducted at four health facilities in Addis Ababa, which were Eka kotebe hospital, millennium hall treatment center, Zewuditu memorial hospital and St Peter TB specialized hospital. Data were collected between December 1 to 31/2020. Health facilities for this study were chosen purposively based on their high number of cases and the major treatment centers found in the city.
One hundred sixty-four (164) clinical nasopharyngeal samples were collected by using 3 ml Miraclean Technology (Shenzhen, China) viral transport media (VTM) from patients who were under investigation for COVID-19 and referred to EPHI for SARS COV-2 testing from December 1 to 30/2020. Nasopharyngeal samples were collected by trained sample collectors and transported to EPHI by triple packaging. Each sample was assigned with unique identification number before nucleic acid extraction. The extraction procedure was carried out immediately upon arrival by using manual and automated extraction methods. Thus, 1.3ml (including 0.8ml dead volume and 0.5ml input volume for extraction) sample was taken from each sample and extracted by Abbott DNA sample preparation system (Abbott Molecular Inc. des Plaines, IL, USA) with a batch of 92 samples and 2 controls (1 positive, 1 negative), and two no-template controls (NTC) were included throughout the procedure (in extraction and detection) of Abbott Real-time SARS-COV-2 (EUA). Similarly, from similar specimen used in automated extraction, 140µl samples was aliquated and used for manual extraction by QIAamp® viral RNA mini kit (QIAGEN GmbH, Hilden, Germany) with a batch of 20 samples and two controls (one negative, one positive), and additionally two NTC were included throughout the procedure (in extraction and detection) of BGI SARS-CoV-2 assay, Daan Gene assay and Sansure Biotech assay.
Automated SARS-CoV-2 viral RNA isolation and purification were done with Abbott DNA sample preparation reagents by the principle of magnetic beads. Sample inactivation and solubilization of viral particles were done with detergent, which contains guanidine iso-thyocyanate for protein denaturation and RNase inactivation. Then, RNA separated from proteins through solid-phase separation using silica; i.e. nucleic acid bind to negatively charged silica (SiO2) is facilitated by guanidinium salts and the basic pH of the lysis buffer. Washing steps could be removed any remnant protein and debris to produce a clear solution. The clear RNA separated from silica-based micro particles are by using the magnetic field of the instrument [15, 16]. On the other hand, manual RNA extraction and purification were performed via spin column method and separation of micro-particle from eluate, are using centrifugation rather than magnetic rack in spin column method .
Abbott Real-time SARS-CoV-2 Assay (EUA)
The Abbott Real-time SARS-CoV-2 assay (Abbott Molecular, Inc.) test was performed as described in the manufacturer instruction. Which has got Emergency Use Authorization from WHO and FDA) [15, 18]. In this protocol pre-extraction sample inactivation was performed with a water bath at 56 oC for 30 minutes , after viral inactivation nucleic acid extraction was done from 0.5 ml VTM on the Abbott m2000 SP instrument and using the Abbott m2000 Sample Preparation System DNA according to the manufacturer’s recommendations. The amplification and detection were performed by Abbott m2000 RT-PCR instrument targeted to dual-target assay for the RdRp and N genes. The SARS-CoV-2 and IC-specific probes are each labeled with a different fluorophore, Carboxyfluorescein (FAM™), Carboxy-X-rhodamine (ROX™), and VIC® P (Proprietary dye) for target and internal control detection, thus allowing for simultaneous detection of both amplified products .
Daan Gene nCoV-2019 assay (EUA)
The Amplification and detection method of this kit is based on the one-step RT-PCR technique. ORF1ab and N genes were selected as the conserved region of Daan Gene technology for amplification and detection of target regions. Specific primers and fluorescent probes are designed (N gene probe is labeled with FAM and ORF1ab probe with VIC) for the detection of SARS-CoV-2 RNA in the specimens. The final eluent and master mix preparation was 5µl of eluate added to 20 µl of master mix for a final volume of 25 µl. Amplification and detection were performed on ABI 7500 RT-PCR instrument simultaneously .
Sansure Biotech detection assay (RUO)
The Sansure Biotech novel coronavirus (2019-nCov) nucleic acid diagnostic kit (PCR-florescence probing) was used for the detection of the ORF1a/b and N gene. The specific probe for each target gene is prepared, the FAM channel is selected for the ORF 1a/b region and the ROX channel is for the N gene. In this detection kit, eluent and master mix addition are as follows; 30µl master mix reagent and 20µl eluted sample were prepared for detection/amplification. ABI 7500 RT-PCR was used for Amplification/detection .
BGI SARS-CoV-2 testing assay (EUA)
BGI SARS-CoV-2 assay is the real-time fluorescent RT-PCR Kit for the diagnosis of COVID-19, a specific target region is found in the ORF1ab region of the SARS-CoV-2 genome. It is using single-gene testing assays. Furthermore, the human housekeeping gene β-actin is the target gene for internal control. The master mixing was done by mixing 20µl master mix reagent and 10µl of the extracted sample RNA to the well pre-filled with PCR-Mix . ABI 7500 RT-PCR instrument was used for amplification and detection.
All nucleic acid amplifications, each assay PCR cycling condition and result interpretation were performed based on respective manufacturer instructions (table-1).
Interpretation of the CRR (reference result)
In this comparative analysis study, we have not used the reference standard method to determine percent agreements (positive, negative and overall) and other comparative parameters of four assay methods. The result was defined by consensus, a similar result which was tested at least with two EUA assays (Abbott SARS-CoV-2 assay and or Daan Gene nCoV-2019 assay and or BGI SARS-CoV-2 assay) by compatible platforms recommended by respective manufacturers was taken as reference result [23, 24].
Data processing and analysis
The data was collected using structured data extraction form, data entry and analysis was done using excel and SPSS version 23.0 statistical software for descriptive statistics. Positive, negative and overall percent agreements were analyzed and Kappa Estimator was employed to determine the strength of agreement of each method with the CRR. Kappa values were interpreted as follows from 0.01–0.20 slight agreement; from 0.21– 0.40 fair agreements, 0.41–0.60 moderate agreement, 0.61–0.80 substantial agreement and 0.81–0.99 perfect agreement .
Ethical clearance was obtained from Addis Ababa University and all experimental protocols of the study were approved by Ethiopian Public Health Institute Scientific ethical review committee. The reference number of EPHI ethical clearance was EPHI/IRB-279-2020. All methods were performed in accordance with the guidelines and regulations of Ethiopian national comprehensive COVID-19 management handbook. Moreover, informed written consent was obtained from all study participants prior to participate in the study.