The comparison of Xpert® Xpress with Cobas® SARS-CoV-2 assay on individual and pooled nasopharyngeal swabs and whole genome sequencing of SARS-CoV-2 samples collected in selected hospitals of Addis Ababa, Ethiopia.
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Date
2023-07
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Addis Ababa University
Abstract
Background: Xpert® Xpress and Cobas® are assays offering rapid test for the detection of
SARS-CoV-2. Pooled testing allows for continued testing even when supplies are relatively
scarce. Whole genome sequencing of SARS-CoV-2 refers to the process of determining the
complete genetic sequence of the virus, providing a detailed and comprehensive analysis of its
entire genome. The agreement between assays for SARS-CoV-2 detection from individual and
pooled samples is uncertain in Ethiopia. Moreover, there is limited availability of whole genome
sequencing data for SARS-CoV-2, leading to insufficient information regarding the circulating
variants, lineages, and mutations.
Objective: The study aimed to compare the performance of Xpert® Xpress with Cobas® SARS-
CoV-2 assays for SARS-CoV-2 detection from individual and pooled nasopharyngeal swabs and
whole genome sequencing of SARS-CoV-2 to identify lineages, sub-lineages and mutations for
nucleocapsid and envelop gene, in Ethiopia.
Methods: A cross-sectional study design was utilized on individuals suspected of having
COVID-19 in seven selected hospitals in Addis Ababa, Ethiopia, between June 25, and July 20,
2022. The samples were tested for SARS-CoV-2 using the GeneXpert® and Cobas 8800®. A
total of fifty samples that tested positive with the Xpert® were chosen and divided into four
groups according to their CT-value and then pooled with negative samples and subjected to
testing using both assays. Positive samples with a CT-value of ≤30 were selected for sequencing.
The CovidSeq Assay was used to prepare sequencing libraries, which were then sequenced on
the Illumina Miseq. Data were entered into Epi data version 3.1 and exported to SPSS version 28
for statistical analysis. Analysis was performed by using descriptive statistics, pearson
correlation, and cohen’s kappa. Nextflow work flow manager and different bioinformatics tools
were used for the analysis of variants, lineages, and mutations.
Results: In this study, a total of 440 nasopharyngeal swabs were collected, and the overall rate of
positivity for SARS-CoV-2 was found to be 100 (22.73%). The overall agreement between the
assays from individual samples was 91.73(95% CI, 89.99% to 92.66 %, κ=0.715) indicating a
substantial level of agreement. The Xpert® assay showed a high positive percent agreement
(98.25%) and negative percent agreement (90.71%). The overall agreement between the assays
from pooled samples was 89%, (95% CI, 86.31% to 91.52% κ=0.552). All 16 sequenced samples
were identified as the Omicron variant, with ten samples assigned to clade 22A. The consensus
XIII
sequence length ranged from 29,578 to 29,903, with a GC% ranging from 33.1% to 39.0%. The
average length of amino acids in the sequences was 9519. The pairwise identity between the
samples and the reference genome ranged from 98.6% to 99.9%. Several amino acid changes
were observed in the sequenced samples, including P13L, P151S, N E31del, N R32del, N
S33del, N P151S, N R203K, N G204R, and N S413R on the N genes. Likewise, there was one
common mutation at position 9 in the E gene, E31del.
Conclusion: The Xpert® performed better than the Cobas® assay in detecting positive results
due to its ability to detect low viral load cases, utilizing the more sensitive nucleocapsid gene
instead of the ORF1a gene used in the Cobas® assay. Confirming presumptive positive results
by different methods and using multiple molecular assays targeting different genes is important
to ensure high sensitivity in detecting RNA. Pooling up to six samples was possible regardless of
viral load and pool size for Xpert®. But viral load effect was significant in all pool size of
Cobas® assay. The agreement on individual samples indicated that both assays can be used
interchangeably for diagnostic purposes. Furthermore, there were variations in amino acid
length, the presence of both unique and existing mutations, and variations in mutation
frequencies among the analyzed sequences.
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Keywords
Agreement, Cobas® SARS-CoV-2 assay, COVID-19, Xpert® Xpress SARS-CoV-2 assay, Pooling, SARS-COV-2, WGS