The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.

Highlights

We developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2Our panel targets nongenic and genic regions in genomic and subgenomic RNAsAll assays detect 1-10 copies and are linear over 3-4 orders of magnitudeAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load AssayClinical samples showed higher copy numbers for targets at the 3' end of the genome.

BACKGROUND

Bedaquiline (BDQ) is recommended for the treatment of multidrug-resistant tuberculosis (MDR TB), however, it has the potential to prolong QTc interval. We assessed the frequency and severity of QTc prolongation in patients receiving BDQ in California.

METHODS

Based on chart review for patients receiving BDQ as part of MDR TB therapy from January 2013-May 2019, we analyzed QTc values at six pre-specified time points during BDQ therapy (baseline, 2, 4, 8, 12, and 24 weeks), as well as peak QTc, time to peak QTc, and the clinical characteristics of patients who had QTc elevation >500 milliseconds (ms) during therapy.

RESULTS

A total of 37 patients were treated with BDQ during the analysis period, with a total of 449 QTc measurements available for analysis. Most patients (89%) received at least one QTc-prolonging drug in addition to BDQ. Median QTc values at all pre-specified time points were <450 ms. Median peak QTc was 455 ms (interquartile range [IQR]: 437-486) and median time to peak was 57 days (IQR: 19-156). Four patients (11%) had a non-transient elevation in QTc to >500 ms, including one patient with profound hypokalemia and one receiving concurrent chemotherapy, but none had cardiac arrhythmia. Less than 10% of patient in our cohort had ECGs performed at all six pre-specified time points.

DISCUSSION

BDQ was generally well-tolerated in a cohort of patients treated for MDR TB in California, with 11% of patients experiencing a non-transient QTc elevation >500 ms, and no episodes of arrhythmia. Frequent ECG monitoring during BDQ therapy presents a challenge for TB clinicians, even in well-resourced countries.