Author: Wong, Chun Wai
Title: Molecular detection and genomic epidemiology of severe acute respiratory syndrome coronavirus 2 in Hong Kong
Advisors: Siu, K. H. Gilman (HTI)
Degree: DHSc
Year: 2022
Subject: Coronavirus infections -- Diagnosis
COVID-19 (Disease) -- Diagnosis
Hong Kong Polytechnic University -- Dissertations
Department: Faculty of Health and Social Sciences
Pages: xvii, 149 pages : color illustrations
Language: English
Abstract: Background:
Real-time reverse transcriptase–polymerase chain reaction assay (RT-PCR) is currently the mainstay for the rapid diagnosis of coronavirus disease 2019 (COVID-19). However, most of the sample-to-answer platforms used for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been validated to test respiratory samples other than nasopharyngeal (NP) samples, such as deep throat saliva (DTS) and specimens collected from the lower respiratory tract (LRT) (e.g., sputum and tracheal aspirate). Nonetheless, these non-validated sample types do have important diagnostic value; therefore, there is a need to validate these sample types for testing using the common RT-PCR platforms. Under Hong Kong Hospital Authority, newly diagnosed COVID-19 cases are confirmed using two methods before issuing a positive report. However, differences in the lower limit of detection and the target genes tested among different RT-PCR assays may give rise to discrepant results, especially when testing samples with low viral loads. Such discrepancies can only be resolved using highly sensitive methods such as digital PCR (dPCR) that allow absolute quantification of the virus. As the COVID-19 pandemic evolved, outbreaks occurred not only in community but also in healthcare settings. Among healthcare workers (HCWs) diagnosed with COVID-19, routine epidemiological investigation might not be adequate to differentiate whether the infection has been acquired from community or nosocomial transmission. To investigate a suspected nosocomial transmission, phylogenomics should be applied to study the genomic epidemiology of SARS-CoV-2 variants detected in patients and HCWs.
This study aimed to (i) evaluate the diagnostic performance of various sample-to-answer platforms in the detection of SARS-CoV-2 from multiple types of respiratory specimens, by using the results of a standard-of-care nucleic acid amplification test (SOC-NAAT) as reference; (ii) apply dPCR to detect SARS-CoV-2 in samples with low viral loads; and (iii) apply phylogenomics to investigate a suspected intra-hospital transmission of SARS-CoV-2.
This study was divided into three phases. Respiratory specimens, including NP, DTS, and LRT specimens, were collected from patients with suspected COVID-19 and first tested by SOC-NAAT. In the first phase, the remnants of samples after testing by SOC-NAAT were retrieved to evaluate the diagnostic performance of three types of sample-to-answer RT-PCR platform for respiratory specimens: (i) Random-access and rapid diagnostic platforms: the Cepheid Xpert Xpress (GeneXpert) SARS-CoV-2 assay and the BioFire Respiratory Panel (RP) 2.1 assay; (ii) Medium-throughput platform: the BD SARS-CoV-2 (BD MAX) assay; and (iii) High-throughput platform: the Hologic Panther Fusion (PF) SARS-CoV-2 assay. Using the composite results of SOC-NAAT as reference, the diagnostic performance of these platforms was verified based on multiple statistical indexes: positive percent agreement, negative percent agreement, Cohen's kappa (k) value, and area under the receiver operating characteristic curve (AUC), to determine whether they are suitable to be adopted in clinical services. In the second phase, leftover RNA samples with low viral loads that showed discrepant results among the different RT-PCR platforms were subjected to absolute quantification by two dPCR platforms: Bio-Rad QX200 ddPCR and Qiagen QIAcuity dPCR. In the final phase, phylogenomics was applied to investigate a suspected intra-hospital transmission of SARS-CoV-2 involving two infected HCWs and nine patients.
Phase 1 study: In terms of their overall agreement with SOC-NAAT, the GeneXpert, BioFire RP 2.1, BD MAX, and PF assays showed k values of 0.98, 0.99, 0.97, and 0.97, respectively, and AUC values of 0.993, 0.997, 0.994, and 0.988 (P < 0.001), respectively.
Phase 2 study: When using droplet dPCR (ddPCR) as the reference method, 25 of the 34 low viral load samples (73.5%) with positive droplet detected, while 8 samples (23.5%) were confirmed as negative by both dPCR platforms. Among these 34 samples, 22 tested negative for the E gene and positive for the N2 gene (E-N+) by the GeneXpert assay; 18 of these 22 samples (81.8%) were confirmed to be positive by ddPCR, while 3 samples were confirmed to be negative by both dPCR platforms.
Phase 3 study: All samples collected from the infected HCWs and patients from a regional hospital with suspected intra-hospital transmission of SARS-CoV-2 were found to belong to the Pangolin lineage B.1.36.27, with an evolutionary rate of 1.93 nucleotide changes per month. The difference in the number of novel single nucleotide polymorphisms between the patients' and HCWs' samples ranged from 3 to 5 nucleotides.
All of the tested RT-PCR assays demonstrated very good agreement with the reference method. Our results showed that these RT-PCR assays are suitable to be implemented in clinical services for testing both validated and non-validated respiratory sample types (DTS and LRT specimens).
dPCR can be used to resolve the discrepant results of low viral load samples obtained by different RT-PCR platforms. Furthermore, most of the E-N+ results obtained by the GeneXpert assay were confirmed to be true positive by QX200 ddPCR. The phylogenetic study of the suspected intra-hospital transmission did not support the occurrence of direct SARS-CoV-2 transmission from patients to HCWs.
Rights: All rights reserved
Access: restricted access

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