Determining SARS-CoV-2 viral infectivity is crucial for patient clinical assessment and isolation decisions. We assessed subgenomic RNA (sgRNA) as a surrogate marker of SARS-CoV-2 infectivity in ...SARS-CoV-2-positive reverse transcription PCR (RT-PCR) respiratory samples (
= 105) in comparison with viral culture as the reference standard for virus replication. sgRNA and viral isolation results were concordant in 99/105 cases (94%), indicating highly significant agreement between the two techniques (Cohen's kappa coefficient 0.88, 95% confidence interval CI 0.78 to 0.97,
< 0.001). sgRNA RT-PCR showed a sensitivity of 97% and a positive predictive value of 94% to detect replication-competent virus, further supporting sgRNA as a surrogate marker of SARS-CoV-2 infectivity. sgRNA RT-PCR is an accurate, rapid, and affordable technique that can overcome culture and cycle threshold (
) value limitations and be routinely implemented in hospital laboratories to detect viral infectivity, which is essential for optimizing patient monitoring, the efficacy of treatments/vaccines, and work reincorporation policies, as well as for safely shortening isolation precautions.
Abstract
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) provides a highly variable cycle threshold (Ct) value that ...cannot distinguish viral infectivity. Subgenomic ribonucleic acid (sgRNA) has been used to monitor active replication. Given the importance of long RT-PCR positivity and the need for work reincorporation and discontinuing isolation, we studied the functionality of normalized viral loads (NVLs) for patient monitoring and sgRNA for viral infectivity detection.
Methods
The NVLs measured through the Nucleocapsid and RNA-dependent-RNA-polymerase genes and sgRNA RT-PCRs were performed in 2 consecutive swabs from 84 healthcare workers.
Results
The NVLs provided similar and accurate quantities of both genes of SARS-CoV-2 at 2 different timepoints of infection, overcoming Ct-value and swab collection variability. Among SARS-CoV-2-positive samples, 51.19% were sgRNA-positive in the 1st RT-PCR and 5.95% in the 2nd RT-PCR. All sgRNA-positive samples had >4 log10 RNA copies/1000 cells, whereas samples with ≤1 log10 NVLs were sgRNA-negative. Although NVLs were positive until 29 days after symptom onset, 84.1% of sgRNA-positive samples were from the first 7 days, which correlated with viral culture viability. Multivariate analyses showed that sgRNA, NVLs, and days of symptoms were significantly associated (P < .001).
Conclusions
The NVLs and sgRNA are 2 rapid accessible techniques that could be easily implemented in routine hospital practice providing a useful proxy for viral infectivity and
coronavirus disease 2019 patient follow-up.
SARS-CoV-2 normalized viral loads and sgRNA detection are 2 rapid accessible tools that overcome Ct value and respiratory sample collection variability. They could be easily implemented in routine hospital practice providing a useful proxy for infectivity and COVID-19 patient follow-up.
Abstract
Background
This study describes the genotypic and phenotypic characterization of novel human cytomegalovirus (HCMV) genetic variants of a cohort of 94 clinically resistant HCMV patients.
...Methods and results
Antiviral-resistant mutations were detected in the UL97, UL54, and UL56 target genes of 25 of 94 (26.6%) patients. The genotype-phenotype correlation study resolved the status of 5 uncharacterized UL54 deoxyribonucleic acid polymerase (G441S, A543V, F460S, R512C, A928T) and 2 UL56 terminase (F345L, P800L) mutations found in clinical isolates. A928T conferred high, triple resistance to ganciclovir, foscarnet, and cidofovir, and A543V had 10-fold reduced susceptibility to cidofovir. Viral growth assays showed G441S, A543V, F345L, and P800L impaired viral growth capacities compared with wild-type AD169 HCMV. Three-dimensional modeling predicted A543V and A928T phenotypes but not R512C, reinforcing the need for individual characterization of mutations by recombinant phenotyping.
Conclusions
Extending mutation databases is crucial to optimize treatments and to improve the assessment of patients with resistant/refractory HCMV infection.
mutations in the
terminase subunit and its associated phenotypes were studied in the context of cytomegalovirus (CMV) transplant recipients clinically resistant to DNA-polymerase inhibitors, naive to ...letermovir. R246C was the only
variant detected by standard and deep sequencing, located within the letermovir-resistance-associated region (residues 230-370). R246C emerged in 2/80 transplant recipients (1 hematopoietic and 1 heart) since first cytomegalovirus replication and responded transiently to various alternative antiviral treatments
. Recombinant phenotyping showed R246C conferred an advanced viral fitness and was sensitive to ganciclovir, cidofovir, foscarnet, maribavir, and letermovir. These results demonstrate a low rate (2.5%) of natural occurring polymorphisms within the letermovir-resistant-associated region before its administration. Identification of high replicative capacity variants in patients not responding to treatment or experiencing relapses could be helpful to guide further therapy and dosing of antiviral molecules.
We provide comprehensive data on the clinical correlates of both CMV genotypic follow-up by standard and deep sequencing and the clinical outcomes, as well as recombinant phenotypic results of this novel mutation. Our study emphasizes that the clinical follow-up in combination with genotypic and phenotypic studies is essential for the assessment and optimization of patients experiencing HCMV relapses or not responding to antiviral therapy. This information may be important for other researchers and clinicians working in the field to improve the care of transplant patients since drug-resistant CMV infections are an important emerging problem even with the new antiviral development.
This observation provides comprehensive data on the clinical correlates of both cytomegalovirus (CMV) genotypic follow-up and clinical monitoring and outcomes for two different solid organ ...transplantation recipients that received letermovir as secondary prophylaxis. Our study emphasizes that monitoring of CMV disease in the patient and early genotypic detection of resistance mutations are essential when using new antiviral drugs for off-label indication in patients experiencing CMV relapses or not responding to standard antiviral therapy. These cases and the bibliography reviewed can be helpful for other researchers and clinicians working in the field to optimize the use of new treatments for transplant recipients since drug-resistant CMV infection is an important emerging problem even with new developments in antiviral treatment.
Abstract
Two transplant recipients (1 kidney and 1 hematopoietic stem cell) received maribavir (MBV) after cytomegalovirus (CMV) infection clinically resistant to standard therapy. Both patients ...achieved CMV DNA clearance within 30 and 18 days; however, the UL97 C480F variant emerged, causing recurrent CMV infection after a cumulative 2 months of MBV and 15 or 4 weeks of ganciclovir treatment, respectively. C480F was not detected under ganciclovir before MBV treatment. Recombinant phenotyping showed that C480F conferred the highest level of MBV resistance and ganciclovir cross-resistance, with impaired viral growth. Clinical follow-up and genotypic and phenotypic studies are essential for the assessment and optimization of patients with suspected MBV resistance.
A cytomegalovirus UL97 C480F mutation emerged after maribavir treatment in 2 transplant recipients (1 kidney, 1 hematopoietic stem cell). Phenotypic assay confirmed that C480F confers a high level of maribavir resistance and ganciclovir cross-resistance, with a decreased replicative capacity.
Cardiac allograft vasculopathy (CAV) remains a major cause of morbidity and mortality among long-term heart transplant recipients. There is an unmet need for a non-invasive biomarker of CAV that ...could obviate the need to perform surveillance coronary angiograms in these patients. Our aim was to evaluate the performance of Donor-derived Cell Free DNA (dd-cfDNA) as a biomarker of CAV.
We prospectively measured dd-cfDNA levels in all patients undergoing routine coronary angiography >1 year after heart transplant at a single center. Endpoints included the association between dd-cfDNA levels and the presence CAV, according to several prespecified criteria.
We included 94 heart transplant recipients, a median of 10.9 years after transplant. Coronary angiogram revealed CAV
, CAV
, CAV
, and CAV
in 61, 19, 14, and 6% of patients, respectively. Comparison of dd-cfDNA levels in patients with CAV
and CAV
(primary end-point) did not show significant differences (0.92%, IQR 0.46-2.0 vs. 0.46%, IQR 0.075-1.5,
= 0.059), nor did the comparison between patients with stable CAV (no new coronary lesions since previous angiogram,
= 77) and progressive CAV (
= 17); dd-cfDNA values 0.735% (IQR 0.195-2.0) vs. 0.9% (IQR 0.12-1.8),
= 0.76. However, we found an association between NTproBNP levels and CAV degree (
= 0.017). Dd-cfDNA levels did not correlate with NTproBNP (ρ = -0.095).
In this study, dd-cfDNA did not perform as a useful biomarker to avoid surveillance coronary angiograms for CAV diagnosis.
Potential Role of Donor-derived Cell Free DNA as a Biomarker in Cardiac Allograft Vasculopathy, NCT04791852.
Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural ...protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (
= 36/111, 32.4%) or prolonged (5 to 20 days) (
= 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the
gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the
E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication.
This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the
pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.
PDF
