Analytical and Clinical Evaluation of a TaqMan Real-Time PCR Assay for the Detection of Chikungunya Virus

Due to the general symptoms presented by the Chikungunya virus(CHIKV)-infected patients, a laboratory test is needed to differentiate CHIKV from other viral infections. The reverse transcription-quantitative real-time PCR (RT-qPCR) is a rapid and sensitive diagnostic tool, and several assays have been developed for detecting and quantifying CHIKV. Since real-time amplification efficiency varies within and between laboratories, an assay must be validated before being used on patient samples. In this study, the diagnostic performance of a TaqMan RT-qPCR assay was evaluated using synthetic RNA and archived patient samples. The cutoff quantification cycle (Cq) value for the assay was determined by experimental evidence. We found the in-house assay was highly sensitive, with a detection limit of 3.95 RNA copies/reaction. The analytical specificity of the assay was 100%. The analytical cutoff Cq value was 37, corresponding to the mean Cq value of the detection limit. Using archived samples characterized previously, the sensitivity and specificity of the assay were 76% and 100%, respectively. The in-house assay was also compared with a commercial assay, and we found that the in-house assay had higher sensitivity. Although further evaluation with prospective patient samples is needed in the future, this validated RT-qPCR was sensitive and specific, which shows its potential to detect CHIKV in clinical sample

Analytical and Clinical Evaluation of a TaqMan Real-Time PCR Assay for the Detection of Chikungunya Virus

ABSTRACT Due to the general symptoms presented by the Chikungunya virus (CHIKV)-infected patients, a laboratory test is needed to differentiate CHIKV from other viral infections. The reverse transcription-quantitative real-time PCR (RT-qPCR) is a rapid and sensitive diagnostic tool, and several assays have been developed for detecting and quantifying CHIKV. Since real-time amplification efficiency varies within and between laboratories, an assay must be validated before being used on patient samples. In this study, the diagnostic performance of a TaqMan RT-qPCR assay was evaluated using synthetic RNA and archived patient samples. The cutoff quantification cycle (Cq) value for the assay was determined by experimental evidence. We found the in-house assay was highly sensitive, with a detection limit of 3.95 RNA copies/reaction. The analytical specificity of the assay was 100%. The analytical cutoff Cq value was 37, corresponding to the mean Cq value of the detection limit. Using archived samples characterized previously, the sensitivity and specificity of the assay were 76% and 100%, respectively. The in-house assay was also compared with a commercial assay, and we found that the in-house assay had higher sensitivity. Although further evaluation with prospective patient samples is needed in the future, this validated RT-qPCR was sensitive and specific, which shows its potential to detect CHIKV in clinical samples. IMPORTANCE Chikungunya virus causes chikungunya fever, a disease characterized by fever, rash, and joint pain. In the early phase of infection, chikungunya fever is always misdiagnosed as other arbovirus infections, such as dengue. Laboratory tests such as RT-qPCR are therefore necessary to confirm CHIKV infection. We evaluated the performance of an in-house RT-qPCR assay, and our study shows that the assay could detect CHIKV in clinical samples. We also show the cutoff determination of the assay, which provides important guidance to scientists or researchers when implementing a new RT-qPCR assay in a laboratory

SARS-CoV-2 genomic surveillance in Malaysia: displacement of B.1.617.2 with AY lineages as the dominant Delta variants and the introduction of Omicron during the fourth epidemic wave

Objectives: This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program. Methods: COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database. Results: From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country. Conclusion: The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness

SARS-CoV-2 genomic surveillance in Malaysia: displacement of B.1.617.2 with AY lineages as the dominant Delta variants and the introduction of Omicron during the fourth epidemic wave

Objectives This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program. Methods COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database. Results From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country. Conclusion The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness