Reverse transcription-polymerase chain reaction (RT-PCR) cycle threshold (Ct) value in detecting the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is inversely proportionate to the virus load in the patient’s specimen. These values could be beneficial in the epidemic trajectory at the population level. The SARS-CoV-2 B.1.617.2 (Delta) variant which emerged in late 2020, caused an unprecedented exponential increase in SARS-CoV-2 infection cases worldwide. In Malaysia, the surge in coronavirus disease 2019 (COVID-19) cases and the inclining positivity rate contributed to the epidemic waves in late May 2021. Sudden surge in cases was suggested to be associated with increased transmission caused by the emergence of the B.1.617.2 variant. In the present study, Ct value distribution of the positive COVID-19 samples from 2020 and 2021 was tabulated against SARS-CoV-2 genomic variants determined from genomic sequencing. A significant decreasing pattern of median Ct values from overall 2020 and 2021 samples was evident (p<0.01). However, notable variability was observed in the Ct values between 2020 and 2021, which samples showing lower median Ct values in 2021. The percentages of SARS-CoV-2 genomic variants B.1.36 and B.1.524 were 31.6% and 68.4%, respectively, for samples obtained in October and December 2020. Whereas samples obtained in June and July 2021 were 100% of the B.1.617.2 variant. The population neutralizing antibody against SARS-CoV-2 during the initial peak of B.1.617.2 was low, however, increased during the B.1.617.2 wave. A decreasing trend in the Ct value distribution from samples tested in our laboratory correlated well with the increasing weekly COVID-19 cases reported by the Malaysia national data, which was subsequently attributed to the emergence of B.1.617.2 variant. This study proposes that analyzing Ct value distribution in screened SARS-CoV-2 samples could reveal population-level transmission dynamics and emerging variants. Coupled with genomic sequencing, it supports early control strategies against new SARS-CoV-2 strains.

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Motile enterococci such as Enterococcus gallinarum has the ability to acquire and transfer antibiotic resistance genes to other enterococci. Even though infections caused by E. gallinarum are rare, the discovery of this bacteria in food sources and in clinical environments is disturbing. Here, we report the isolation and identification of E. gallinarum from the wound of a hospital in-patient. The isolate was identified using 16S rDNA sequencing. Isolate 146 harboured the vanA and vanC1 gene clusters, was vancomycin-susceptible, and displayed resistance to ampicillin, penicillin, erythromycin and teicoplanin. This isolate also showed intermediate resistance to linezolid and sequencing of the 23S rRNA peptidyl transferase region did not unveil any known mutations associated to the conferment of linezolid resistance. The presence of vanA did not confer resistance to vancomycin. Structural analyses into the Tn1546 transposon carrying the vanA gene revealed distinct genetic variations in the vanS, vanY and vanS-vanH intergenic region that could be associated to the atypical antibiotic resistance phenotypes of isolate 146. Finding from this study are suggestive of the occurrence of interspecies horizontal gene transfer and that similarities in genotypic characteristic may not necessarily correlate with actual antibiotic resistance pattern of E. gallinarum.