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Since the 2019 coronavirus disease (COVID-19) outbreak, many commercial nucleic acid amplification tests (NAATs) have been developed around the world and have become standard assays. Although several tests were quickly developed and applied to laboratory diagnostic tests, the performance of these tests has not been evaluated in a variety of settings. Therefore, this study aimed to evaluate the performance of the Abbott SARS-CoV-2, Daan Gene, BGI, and Sansure Biotech assays using the Composite Reference Standard (CRS). The study was conducted at the Ethiopian Public Health Institute (EPHI) from 1 to 30 December 2020. 164 nasopharyngeal samples were extracted using the QIAamp RNA mini kit and the Abbott DNA sample preparation system. Of 164 specimens, 59.1% were positive and 40.9% were negative for CRS. Sansure Biotech positivity was significantly low compared to CRS (p < 0.05). Sansure Biotech positivity was significantly low compared to CRS (p < 0.05). Положительные результаты Sansure Biotech были значительно ниже по сравнению с CRS (p < 0,05). Sansure Biotech’s positive results were significantly lower compared to CRS (p < 0.05).与CRS 相比，Sansure Biotech 的阳性率显着较低（p < 0.05）。与CRS 相比，Sansure Biotech 的阳性率显着较低（p < 0.05）。 У Sansure Biotech было значительно меньше положительных результатов по сравнению с CRS (p < 0,05). Sansure Biotech had significantly fewer positive results compared to CRS (p < 0.05). The overall agreement of the four analyzes was 96.3–100% compared to CRS. In addition to the low positivity rate of the Sansure Biotech assay, the performance of the four assays was nearly comparable. As such, the Sansure Biotech [Research Only (RUO)] assay requires additional validation for its use in Ethiopia. Finally, additional research should be considered to evaluate assays with appropriate manufacturer’s claims.
Laboratory testing is part of the World Health Organization (WHO) Strategic Plan for Coronavirus Disease 2019 (COVID-19) Preparedness and Response (SPRP). WHO advises that countries need to build laboratory capacity to improve preparedness, proper case management, vigilance and rapid response to public health challenges. This suggests that the role of the laboratory is key to characterizing the disease and epidemiology of emerging infectious agents and controlling their spread.
The diagnosis of COVID-19 requires epidemiological and medical information, personal symptoms/signs, and radiographic and laboratory data2. Since the COVID-19 outbreak was reported in Wuhan, China, many commercial nucleic acid amplification tests (NAATs) have been developed around the world. Real-time reverse transcription polymerase chain reaction (rRT-PCR) has been used as a routine and standard method for laboratory diagnosis of severe acute respiratory syndrome 2 (SARS-CoV-2)3 infection. Molecular detection of SARS-CoV-2 is typically based on the N (nucleocapsid protein gene), E (envelope protein gene), and RdRp (RNA-dependent RNA polymerase gene) genes in ORF1a/b (open reading frame 1a/b). gene) region identified from the viral genome. They are considered to be the main conserved regions found in viral genomes for virus recognition4. Among these genes, the RdRp and E genes have high analytical detection sensitivity, while the N gene has low analytical sensitivity5.
The performance of PCR assays may vary depending on various factors such as: extraction reagents, amplification/detection reagents, extraction method, quality of the PCR machine and other instruments. As of April 2020, more than 48 different diagnostic devices from nine countries have received Emergency Use Authorization (EUA) for COVID-196 diagnostics. In Ethiopia, more than 14 real-time PCR platforms are used for PCR detection of SARS-CoV-2 at 26 public health institutions, including ABI 7500, Abbott m2000, Roche 48000 and Quant-studio7. In addition, various PCR test kits are available, such as Daan Gene test, Abbott SARS-CoV-2 test, Sansure Biotech test, and SARS-CoV-2 BGI test. Although rRT-PCR is highly sensitive, some patients with COVID-19 report false negative results due to insufficient copies of viral ribonucleic acid (RNA) in samples due to improper collection, transport, storage and handling, and laboratory testing. conditions and actions of personnel8. In addition, sample or control mishandling, cycle threshold (Ct) setting, and cross-reactivity with other pathogenic nucleic acids or inactive/residual SARS-CoV-2 RNA can lead to false positive results in rRT-PCR9 assays. Thus, it is clear that PCR tests can indeed identify carriers of gene fragments, as they cannot even distinguish between truly active viral genes, so the tests can only identify carriers and not patients10. Therefore, it is important to assess diagnostic performance using standard methods in our setting. Although many NAAT reagents are available at the Ethiopian Public Health Institute (EPHI) and throughout the country, no comparative evaluation of their effectiveness has yet been reported. Therefore, this study aimed to evaluate the comparative performance of commercially available kits for the detection of SARS-CoV-2 by rRT-PCR using clinical specimens.
A total of 164 participants with suspected COVID-19 were included in this study. The majority of samples were from treatment centers (118/164 = 72%), while the remaining 46 (28%) participants were from non-treatment centers. Among participants not treated at the center, 15 (9.1%) had clinically suspected cases and 31 (18.9%) had contacts of confirmed cases. Ninety-three (56.7%) participants were male, and the mean (± SD) age of the participants was 31.10 (± 11.82) years.
In this study, positive and negative rates of four tests for COVID-19 were determined. Thus, the positive rates of the Abbott SARS-CoV-2 assay, Daan Gene 2019-nCoV assay, SARS-CoV-2 BGI assay, and Sansure Biotech 2019-nCoV assay were 59.1%, 58.5%, 57.9% and 55.5% respectively. The positive and negative composite reference standard (CRS) scores were 97 (59.1%) and 67 (40.9%), respectively (Table 1). In this study, the definition of CRS was based on the “any positive” rule, whereby out of four test results, two or more test results that gave the same result were considered true positive or negative.
In this study, we found a negative percentage agreement (NPA) of 100% (95% CI 94.6–100) for all analyzes compared to CRS. The Sansure Biotechnology analysis showed a minimal PPA of 93.8% (95% CI 87.2-97.1) and the Daan Gene 2019-nCoV analysis had an overall agreement of 99.4% (95% CI 96.6-99.9). In contrast, overall agreement between the SARS-CoV-2 BGI assay and the Sansure Biotech 2019-nCoV assay was 98.8% and 96.3%, respectively (Table 2).
Cohen’s kappa coefficient of agreement between CRS and Abbott SARS-CoV-2 assay results was fully consistent (K = 1.00). Similarly, Cohen’s kappa values detected by Daan Gene 2019-nCoV, SARS-CoV-2 BGI, and Sansure Biotech 2019-nCoV are also fully consistent with CRS (K ≥ 0.925). In this comparative analysis, the chi-square test (McNemar test) showed that the Sansure Biotech 2019-nCoV assay results were significantly different from the CRS results (p = 0.031) (Table 2).
As shown in Fig. 1 the percentage of lowest Ct value (< 20 Ct) of Abbott SARS-CoV-2 assay (combined RdRp and N gene) was 87.6% and ORF1a/b gene Ct value of Sansure Biotech 2019-nCoV assay showed that the percentage of low Ct value (< 20 Ct) was 50.3% and the high Ct value (36–40 Ct) was 3.2%. 1 the percentage of lowest Ct value (< 20 Ct) of Abbott SARS-CoV-2 assay (combined RdRp and N gene) was 87.6% and ORF1a/b gene Ct value of Sansure Biotech 2019-nCoV assay showed that the percentage of low Ct value (< 20 Ct) was 50.3% and the high Ct value (36–40 Ct) was 3.2%. As shown in Fig. 1, процент наименьшего значения Ct (< 20 Ct) анализа Abbott SARS-CoV-2 (комбинированный ген RdRp и N) составил 87,6%, а значение Ct гена ORF1a/b анализа Sansure Biotech 2019-nCoV показало что процент низкого значения Ct (< 20 Ct) составлял 50,3%, а высокое значение Ct (36–40 Ct) составляло 3,2%. 1, the percentage of the lowest Ct value (< 20 Ct) analysis of Abbott SARS-CoV-2 (combined gene RdRp and N) was 87.6%, and the Ct value of ORF1a/b gene analysis of Sansure Biotech 2019-nCoV showed that the percentage of low Ct value (< 20 Ct) accounted for 50.3%, and high value Ct (36–40 Ct) accounted for 3.2%.如图1 所示，Abbott SARS-CoV-2 检测（结合RdRp 和N 基因）的最低Ct 值百分比（< 20 Ct）为87.6%，Sansure Biotech 2019-nCoV 检测的ORF1a/b 基因Ct 值显示低Ct 值(< 20 Ct) 的百分比为50.3%，高Ct 值(36–40 Ct) 的百分比为3.2%。 As shown in Figure 1, the lowest Ct value percentage (< 20 Ct) of Abbott SARS-CoV-2 test (combination of RdRp and N gene) is 87.6%, the ORF1a/b gene Ct value of Sansure Biotech 2019-nCoV test shows low Ct值(< 20 Ct) 的 percentage is 50.3%, 高Ct 值(36–40 Ct) 的 percentage is 3.2%. Как показано на рисунке 1, анализ Abbott SARS-CoV-2 (сочетающий гены RdRp и N) имел самое низкое процентное значение Ct (< 20 Ct) в размере 87,6%, а значение Ct гена ORF1a/b в исследовании Sansure Biotech 2019- Анализ nCoV показал низкий Ct. As shown in Figure 1, the Abbott SARS-CoV-2 assay (combining the RdRp and N genes) had the lowest percentage Ct value (< 20 Ct) at 87.6%, while the Ct value of the ORF1a/b gene in the Sansure Biotech 2019 study – The analysis of nCoV showed a low Ct. Процент значений (< 20 Ct) составил 50,3%, а процент высоких значений Ct (36–40 Ct) составил 3,2%. The percentage of values (< 20 Ct) was 50.3%, and the percentage of high Ct values (36–40 Ct) was 3.2%. The Abbott SARS-CoV-2 B test recorded Ct values above 30. On the other hand, on the BGI SARS-CoV-2 assay ORF1a/b gene had a high Ct value (> 36 Ct) percentage was 4% (Fig. 1). On the other hand, on the BGI SARS-CoV-2 assay ORF1a/b gene had a high Ct value (> 36 Ct) percentage was 4% (Fig. 1). С другой стороны, в анализе BGI SARS-CoV-2 ген ORF1a/b имел высокое значение Ct (> 36 Ct), процент которого составлял 4% (рис. 1). On the other hand, in the analysis of BGI SARS-CoV-2 gene ORF1a/b had a high Ct value (> 36 Ct), the percentage of which was 4% (Fig. 1).另一方面，在BGI SARS-CoV-2 检测中，ORF1a/b 基因具有高Ct 值（> 36 Ct）的百分比为4%（图1）。 On the other hand, in BGI SARS-CoV-2 detection, the percentage of ORF1a/b gene with high Ct value (>36 Ct) is 4% (Figure 1). С другой стороны, в анализе BGI SARS-CoV-2 процент генов ORF1a/b с высокими значениями Ct (>36 Ct) составил 4% (рис. 1). On the other hand, in the BGI SARS-CoV-2 analysis, the percentage of ORF1a/b genes with high Ct values (>36 Ct) was 4% (Fig. 1).
In this study, we took 164 nasopharyngeal samples. For all types of assays, RNA isolation and amplification was performed using the methods and kits recommended by the respective manufacturers.
This study demonstrated that Abbott’s test for SARS-CoV-2 has the same detection performance as CRS, with 100% positive, negative, and overall concordance. Cohen’s kappa agreement is 1.00, indicating full agreement with CRS. A similar study by the University of Washington in the US found that the overall sensitivity and specificity of the Abbott test for SARS-CoV-2 was 93% and 100%, respectively, compared to the laboratory-determined assay (LDA) of the CDC. 11. The Abbott SARS-CoV-2 detection system is based on the simultaneous combined detection of the N and RdRp genes, as both genes are more sensitive, minimizing false negatives12. A study in Vienna, Austria also showed that large extraction sample volumes and detection eluent volumes minimized dilution effects and increased detection efficiency13. Thus, Abbott’s perfect match for the SARS-CoV-2 assay can be associated with a platform detection system that simultaneously detects combinatorial genes, extracts a large number of samples (0.5 ml), and uses a large amount of eluent (40 µl).
Our results also showed that the detection performance of the Daan genetic test was almost the same as that of CRS. This is consistent with a study14 conducted at Anhui University in Huainan, China, and the manufacturer’s claim of 100% positive agreement. Despite reports of consistent results, one sample was false negative after retesting the same eluate, but was positive in the Abbott SARS-CoV-2 and Sansure Biotech nCoV-2019 assays. This suggests that there may be variability in results across different types of assays. Nevertheless, in the study carried out in China15, the result of the Daan Gene assay was significantly different (p < 0.05) compared to their lab-defined reference assay. Nevertheless, in the study carried out in China15, the result of the Daan Gene assay was significantly different (p < 0.05) compared to their lab-defined reference assay. Тем не менее, в исследовании, проведенном в Китае15, результат анализа Daan Gene значительно отличался (p < 0,05) от их лабораторного эталонного анализа. However, in a study in China15, Daan Gene’s analysis result was significantly different (p < 0.05) from their laboratory reference analysis.然而，在中国进行的研究中15，大安基因检测的结果与其实验室定义的参考检测相比有显着差异（p < 0.05）。然而，在中国进行的研究中15，大安基因检测的结果与其实验室定义的参考检测相比有显着差 <0.05 Однако в исследовании, проведенном в Китае15, результаты генетического теста Daan значительно отличались (p < 0,05) по сравнению с его эталонным лабораторным тестом. However, in a study in China15, the results of Daan’s genetic test were significantly different (p < 0.05) compared to its reference laboratory test. This discrepancy may be due to the sensitivity of the reference test to detect SARS-CoV-2, and further studies may be important to determine the cause.
In addition, our study evaluated the comparative performance of the SARS-CoV-2 BGI assay with CRS, showing excellent positive percentage agreement (PPA = 97.9%), negative percentage agreement (NPA = 100%), and overall percentage agreement by gender (OPA). ). = 98.8%). Cohen’s Kappa values showed good agreement (K = 0.975). Studies in the Netherlands16 and China15 have shown consistent results. The SARS-CoV-2 BGI test is a single gene (ORF1a/b) detection test using 10 µl amplification/detection eluate. Despite good statistical agreement with our reference results, the analysis missed two positive samples (1.22%) of the total sample. This can have huge clinical implications for transmission dynamics at both the patient and community levels.
Another comparative analysis included in this study was the Sansure Biotech nCoV-2019 rRT-PCR (RUO) assay; the overall match percentage was 96.3%. The strength of agreement was also determined by the Cohen’s Kappa value, which was 0.925, indicating full agreement with the CRS. Again, our results are identical to studies conducted at Central South University in Changsha, China, and at the Clinical Laboratory Department of Liuzhou People’s Hospital, Liuzhou City, China17. Even though the above good statistical concordance was recorded, the chi-square test (MacNemar test) showed that the result of the Sansure Biotech assay has had a statistically significant difference compared to CRS (p < 0.005). Even though the above good statistical concordance was recorded, the chi-square test (MacNemar test) showed that the result of the Sansure Biotech assay has had a statistically significant difference compared to CRS (p < 0.005). Несмотря на то, что было зафиксировано указанное выше хорошее статистическое соответствие, критерий хи-квадрат (критерий Макнемара) показал, что результат анализа Sansure Biotech имеет статистически значимое различие по сравнению с CRS (p < 0,005). Although the good statistical agreement above was recorded, the chi-square test (McNemar test) showed that the result of the Sansure Biotech assay had a statistically significant difference compared to the CRS (p < 0.005).尽管记录了上述良好的统计一致性，但卡方检验（MacNemar 检验）表明，Sansure Biotech 检测的结果与CRS 相比具有统计学显着差异（p < 0.005）。尽管 记录 了 上述 良好 统计 一致性 ， 但 检验 （（macnemar 检验 表明 ， ， sansure biotech 检测 结果 与 crs 相比 具有 显着 （（p <0.005。。。。。。。。。。。。。。。。。。。）））） Несмотря на отмеченное выше хорошее статистическое соответствие, критерий хи-квадрат (критерий Макнемара) показал статистически значимую разницу (p < 0,005) между анализом Sansure Biotech и CRS. Despite the good statistical agreement noted above, the chi-square test (McNemar test) showed a statistically significant difference (p < 0.005) between the Sansure Biotech assay and the CRS. Six samples (3.66%) were found to be false negatives compared to CRS (Supplementary Table 1); this is very important, especially given the dynamics of transmission of the virus. The above data also supports this low detection rate15.
In this study, Ct values were determined for each assay and respective platform, with the lowest mean Ct value reported in the Abbott SARS-CoV-2 assay. This result may be related to Abbott’s simultaneous combined genetic testing system for the detection of SARS-CoV-2. Therefore, according to Figure 1, 87.6% of Abbott SARS-CoV-2 results had Ct values below 20. Only a small number of sample results (12.4%) were in the 20-30 range. Ct values above 30 were not recorded. In addition to Abbott’s use of the SARS-CoV-2 panel genetic testing format, this result may be related to the lower detection limit (32.5 RNA copies/mL)18, which is three times lower than the company’s lower limit of 100 RNA copies/mL. ml)19.
This study has some limitations: firstly, we do not have standard/reference methods [such as viral load or other laboratory tests (LDA)] due to lack of resources. Second, all specimens used in this study were nasopharyngeal swabs, while the results were not applicable to other specimen types, and third, our sample size was small.
This study compared the performance of four rRT-PCR assays for SARS-CoV-2 using nasopharyngeal samples. All detection assays had nearly comparable performance, with the exception of the Sansure Biotech assay. Besides, the low positivity rate was identified in the Sansure Biotech assay compared to the CRS (p < 0.05). Besides, the low positivity rate was identified in the Sansure Biotech assay compared to the CRS (p < 0.05). Кроме того, в тесте Sansure Biotech был выявлен низкий процент положительных результатов по сравнению с CRS (p < 0,05). In addition, the Sansure Biotech test showed a low percentage of positive results compared to CRS (p < 0.05).此外，与CRS 相比，Sansure Biotech 检测的阳性率较低(p < 0.05)。此外，与CRS 相比，Sansure Biotech 检测的阳性率较低(p < 0.05)。 Кроме того, анализ Sansure Biotech имел более низкий уровень положительных результатов по сравнению с CRS (p < 0,05). In addition, the Sansure Biotech assay had a lower positivity rate compared to CRS (p < 0.05). The Sansure Biotech nCoV-2019 (RUO) analysis of PPA, NPA and overall agreement exceeded 93.5% with a Cohen Kappa strength of agreement value of 0.925. Finally, the Sansure Biotech Assay (RUO) needs further validation for use in Ethiopia, and additional research should be considered to evaluate claims from individual manufacturers.
Comparative study design was conducted at four health facilities in Addis Ababa, Eka Kotebe Hospital, Millennium Church Treatment Centre, Zewooditu Memorial Hospital, and St. Peter’s Tuberculosis Specialist Hospital. The data was collected between December 1 and 31, 2020. The medical facilities for this study were purposefully chosen based on their high number of cases and the availability of major treatment centers in the city. Similarly, instruments, including the ABI 7500 and Abbott m2000 real-time PCR instruments, were selected according to the recommendations of the NAAT reagent manufacturers, and four PCR detection kits were selected for this study, as most laboratories in Ethiopia used at least at least four of them. Gene test, Abbott SARS-CoV-2 test, Sansure Biotech test, and SARS-CoV-2 BGI test performed during the study).
Testing for SARS-CoV-2 was performed from 1 to 30 December 2020 using 3 ml of Viral Transport Medium (VTM) (Miraclean Technology, Shenzhen, China) from individuals under investigation for COVID-19 referred to EPHI. Nasopharyngeal samples were collected by trained sample collectors and sent to EPHI in triple packs. Prior to nucleic acid isolation, each sample is assigned a unique identification number. Extraction is performed from each sample immediately upon arrival using manual and automatic extraction methods. Thus, for the automatic extraction of Abbott m2000, 1.3 ml (including 0.8 ml dead volume and 0.5 ml extraction inlet volume) of the sample was extracted from each sample and passed through the Abbott DNA Sample Preparation System (Abbott Molecular Inc. des Plaines, IL, USA). ) A batch of 96 [92 samples, two detection controls and two non-template controls (NTC)] was included in the overall process (retrieval and detection) of two rounds of SARS-CoV-2 (EUA) in real time. mining. Similarly, for manual extraction, use the same samples (for automatic extraction and discovery). Thus, throughout the process, 140 µl samples were aliquoted and extracted using the QIAamp Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany) in batches of 24 (including 20 samples, two assay controls and two NTCs) over nine rounds. Manually extracted eluates were amplified and detected using an ABI 7500 thermal cycler using SARS-CoV-2 BGI assay, Daan Gene assay, and Sansure Biotech assay.
Automated isolation and purification of SARS-CoV-2 viral RNA follows the magnetic bead principle using Abbott DNA sample preparation reagents. Inactivation of samples and solubilization of viral particles is carried out using a detergent containing guanidine isothiocyanate to denature the protein and inactivate RNase. The RNA is then separated from the protein by solid phase separation using silica, i.e. the guanidinium salt and the alkaline pH of the lysis buffer promote binding of the nucleic acids to the silica (SiO2). The rinsing step removes remaining proteins and debris to produce a clear solution. Transparent RNA is isolated from silica-based microparticles using the instrument’s magnetic field20,21. On the other hand, manual isolation and purification of RNA is carried out by the spin column method using centrifugation instead of a magnetic stand and separation of microparticles from the eluent.
The Abbott Real-Time SARS-CoV-2 Detection Test (Abbott Molecular, Inc.) was performed according to the manufacturer’s instructions, which received EUA19,22 from the WHO and FDA. In this protocol, sample inactivation before extraction was performed in a water bath at 56 °C for 30 min. After virus inactivation, nucleic acid extraction was performed on an Abbott m2000 SP instrument from 0.5 ml VTM using an Abbott m2000 DNA sample preparation system. according to the manufacturer. Amplification and detection were performed using an Abbott m2000 RT-PCR instrument, and dual detection was performed for the RdRp and N genes. ROX) and VIC P (proprietary dye) for targeting and detection of internal controls, allowing simultaneous detection of both amplification products 19 .
The amplification detection method of this kit is based on one-step RT-PCR technology. The ORF1a/b and N genes were selected as conserved regions by Daan Gene Technology to detect target region amplification. Specific primers and fluorescent probes (N gene probes labeled with FAM, ORF1a/b probes labeled with VIC) have been designed to detect SARS-CoV-2 RNA in samples. The final eluent and master mixes were prepared by adding 5 µl of eluent to 20 µl of the master mix to a final volume of 25 µl. Amplification and detection were performed simultaneously on an ABI 750024 real-time PCR instrument.
The ORF1a/b and N genes were detected using the Sansure Biotech nCoV-2019 Nucleic Acid Diagnostic Kit (fluorescent PCR detection). Prepare specific probes for each target gene by selecting the FAM channel for the ORF1a/b region and the ROX channel for the N gene. To this assay kit, eluent and master mix reagents are added as follows: prepare 30 µl master mix reagent and 20 µl eluted sample for detection/amplification. Real-time PCR ABI 750025 was used for amplification/detection.
The SARS-CoV-2 BGI test is a fluorescent real-time rRT-PCR kit for the diagnosis of COVID-19. The target region is located in the ORF1a/b region of the SARS-CoV-2 genome, which is a single gene detection method. In addition, the human housekeeping gene β-actin is an internally regulated target gene. The master mix is prepared by mixing 20 µl of the master mix reagent and 10 µl of the extracted RNA sample in a well plate26. An ABI 7500 fluorescent quantitative real-time PCR instrument was used for amplification and detection. All nucleic acid amplification, PCR run conditions for each assay, and interpretation of results were performed according to the respective manufacturer’s instructions (Table 3).
In this comparative analysis, we did not use the reference standard method to determine percent agreement (positive, negative, and overall) and other comparison parameters for the four analyses. Each test comparison was done with CRS, in this study the CRS was set by the rule “any positive” and the result was determined, not by a single test, we used at least two matched test results. In addition, in the case of COVID-19 transmission, false negative results are more dangerous than false positive results. Therefore, to say “positive” as accurately as possible from a CRS result, at least two assay tests must be positive, meaning that at least one positive result is likely to come from an EUA assay. Thus, out of four test results, two or more test results that give the same result are considered true positive or negative18,27.
Data was collected using structured data extraction forms, data entry and analysis were performed using Excel statistical software and SPSS version 23.0 for descriptive statistics. Positive, negative, and overall percent agreement were analyzed, and a Kappa score was used to determine the degree of agreement of each method with CRS. Kappa values are interpreted as follows: 0.01 to 0.20 for mild agreement, 0.21 to 0.40 for general agreement, 0.41-0.60 for moderate agreement, 0.61-0.80 for major agreement and 0.81-0.99 for complete agreement28.
Ethical clearance was obtained from the University of Addis Ababa and all experimental protocols for this study were approved by the Ethiopian Public Health Institute’s Scientific Ethics Review Board. The reference number for the EPHI Ethics License is EPHI/IRB-279-2020. All methods were applied in accordance with the recommendations and provisions of the Ethiopian National Comprehensive Guidelines for the Treatment of COVID-19. In addition, written informed consent was obtained from all study participants prior to participation in the study.
All data obtained or analyzed in this study are included in this published article. Data supporting the results of this study are available from the respective author upon reasonable request.
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