FormalPara Key Summary Points

Subgenomic RNA (sgRNA) may have clinical utility to help monitor virologic response more accurately than genomic RNA (gRNA) in kidney transplant recipients who receive remdesivir.

Patients with negative sgRNA at baseline may not require antiviral treatment.

Patients with positive sgRNA at day 5 could benefit from prolonged or combined therapies.

Introduction

Patients, such as kidney transplant recipients (KTR), receiving immunosuppressive drugs are among the most vulnerable groups to present severe SARS-CoV-2 infection, with high mortality and morbidity rates reported [1, 2]. One explanation for these worse outcomes is that immunosuppressed patients present prolonged shedding and impaired viral clearance, making clinical management challenging [3,4,5].

Remdesivir (RDV) has potent in vitro activity against all SARS-CoV-2 variants, and has shown rapid clearance of viable virus from respiratory samples [6,7,8]. Several studies have reported that early administration of RDV is associated with a reduced length of hospital stay and mortality rates [9,10,11].

Reverse transcription–polymerase chain reaction (RT-PCR) is the most sensitive and widely used technique for the diagnosis of SARS-CoV-2. However, while RT-PCR is able to detect the presence of viral genomic RNA (gRNA), it is not able to distinguish between viable, replicating, or nonviable virus, which can remain positive in respiratory secretions for weeks due to the detection of viral particle debris. On the other hand, although the cycle threshold (Ct) value provides a semi-quantitative estimate of the viral load, Ct values have some limitations as they present high variability in respiratory samples and in several other factors, such as analytical assay sensitivity and specimen quality. Virus isolation is the gold standard for determining virus infectivity, but it is labor-intensive and has some limitations, making this method challenging for many laboratories [12]. Therefore, additional surrogate markers of viral viability are crucial to identify and manage patients with active infection. Early in the pandemic, Wölfel et al. [13] showed good correlation between qualitative subgenomic RNA [14] (sgRNA) and viral cultures. sgRNA is only transcribed in infected cells and is poorly packaged into virions, suggesting the presence of active replication. More recently, our microbiology laboratory confirmed the good correlation between these two parameters in more than 100 samples [15], suggesting that sgRNA may be a good parameter to identify patients who require treatment with RDV and to evaluate their response to antiviral treatment.

The aim of this study was to evaluate the dynamics of SARS-CoV-2 sgRNA in naso-pharyngeal swabs (NPS) of KTR with COVID-19 before and after treatment with RDV compared to gRNA RT-PCR and its use as a surrogate marker of viral replication.

Methods

Patients

We conducted a retrospective study at a tertiary university referral hospital with an active kidney transplantation program (annual average: 165 kidney transplants), in Barcelona, Spain. We analyzed KTR who required administered RDV therapy for SARS-CoV-2 infection from November 2021 to February 2022. All patients provided signed informed consent. The Ethics Committee of our institution approved the study (HCB/2022/0072).

Variables and Outcomes

The variables collected included: age, gender, co-morbidities, prior transplants, simultaneous transplants, induction immunosuppressive regimen, maintenance immunosuppressive regimen, incidence of biopsy-proven acute allograft rejection (6 months prior to COVID-19), time from transplantation to SARS-CoV-2 infection, lymphopenia prior to SARS-CoV-2 infection (≤ 1000/mcL), SARS-CoV-2 vaccination status, severity of SARS-CoV-2 infection, other concomitant therapies against SARS-CoV-2 infection, and outcomes in terms of hospital admission, intensive care unit (ICU) admission, and death at 30 days. The main outcomes were discharge from the hospital within the first 6 days after admission (median value of hospital admission in the total population included in the study) and all-cause mortality at 30 days. We defined patients as having asymptomatic infection when testing positive for SARS-CoV-2, but having no symptoms consistent with COVID-19, mild infection if they had any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell), but did not have shortness of breath, dyspnea, or abnormal chest imaging.

Moderate infection was defined if there was evidence of lower respiratory disease during clinical assessment or imaging and who had an oxygen saturation measured by pulse oximetry (SpO2) ≥ 94% on room air at sea level. Finally, severe infection was present if they presented SpO2 < 94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) < 300 mm Hg, a respiratory rate > 30 breaths/min, or lung infiltrates > 50%.

Microbiological Methods

All NPS (pre- and post-RVD) were frozen at – 80 °C after RT-PCR of gRNA for diagnosis of infection, and RT-PCR of sgRNA was performed after enrolment of all the patients.

The presence of SARS-CoV-2 gRNA was determined by RT-PCR in the Cobas® 6800 automated system (Roche, Switzerland) according to the manufacturer's instructions. For the detection of SARS-CoV-2 sgRNA, total sample nucleic acid was extracted using MagNA Pure Compact (Roche). All the samples were tested for the presence of envelope (E) sgRNA using the leader-specific primer described by Wölfel et al. [13], as well as primers and probes targeting sequences downstream of the E gene start codons [16]. RT-PCR was performed using the RNA process control kit (Roche) in the LightCycler® 480 thermal cycler system (Roche). This system provides an internal control to verify process optimization from the beginning of the extraction process to the final RT-PCR result. All Ct values > 39 were considered negative for gRNA and sgRNA according to the manufacturer's instructions.

Statistical Analysis

Continuous variables were described as mean with standard deviation (SD) or median and interquartile range (IQR) according to data distribution. Categorical variables were described as either absolute frequencies or percentages. We used the Fisher’s exact test to compare early discharge according to the sgRNA result after treatment . Univariate analysis was performed to analyze factors associated with positive sgRNA post-RDV treatment in the population with a positive sgRNA at admission. Age, sex, and the variables associated with the primary outcome with a p < 0.1 were finally entered into the multivariable logistic analysis. All statistical tests were conducted with a 95% confidence interval, and a p value < 0.05 was considered significant. The software SPSS v.25 (SPSS, Chicago, IL, US) was used to perform the analyses.

Results

During the study period, 34 patients with positive gRNA in the NPS before and after RDV treatment were included. Table 1 shows the baseline characteristics of the patients included according to COVID-19 severity. The median time from kidney transplantation was 80 months (IQR 3–321) and 6 patients presented SARS-CoV-2 infection during the first year after transplantation. Nine patients (26%) presented lymphopenia prior to SARS-CoV-2 infection. The majority of patients received 3 or more SARS-CoV-2 vaccine doses (76%), and the median time between symptom onset and RDV treatment was 5 days (IQR3–8.5). RDV was administered as a single dose of 200 mg, followed by 2–4 more daily doses of 100 mg, without adjustment for renal function. Thirty-two (94%) patients received 5 doses of RDV.

Table 1 Baseline characteristics of 34 kidney transplant recipients treated with remdesivir according to SARS-CoV-2 severity
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The majority of patients (73%) included presented moderate SAR-CoV-2 disease, while 17% presented severe and 8% mild disease (8%). Dexamethasone was the most common concomitant therapy in 34% of the patients. Seven patients required ICU admission (21%). Three patients (9%) required hospital readmission within 30 days after diagnosis) and 3 patients (9%) died within 30 days. The median number of days of hospitalization was 6 days (IQR 2–112).

The median Ct values of gRNA were 20 at baseline (IQR 13–28) and 28 after RDV treatment (IQR 10–36). A comparison of Ct values of gRNA and sgRNA at baseline and after 5 days of remdesivir treatment according to SARS-CoV-2 severity is depicted in Table 2. Median Ct values of gRNA and sgRNA at day 5 after remdesivir therapy were higher in patients presenting with mild or moderate disease compared with those with severe disease.

Table 2 Comparison of Ct values of genomic RNA (gRNA) and subgenomic RNA (sgRNA) at baseline and after 5 days of remdesivir treatment according to SARS-CoV-2 severity
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A flowchart of outcomes according to sgRNA results is shown in Fig. 1.

Fig. 1
figure 1

Flowchart of outcomes according to sgRNA results

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Baseline Negative sgRNA

Baseline sgRNA was negative in 5 patients (15%) and remained negative after treatment in all patients except one who switched to positive within 24 h of the first test. In the sgRNA negative group, all patients were discharged from hospital in ≤ 10 days and no patient died.

Baseline Positive sgRNA

Twenty-nine (85%) patients had positive sgRNA at baseline. Of these, 15 patients (52%) presented negative sgRNA after RDV treatment. All the patients in this group had favorable outcomes, with most (80%) being discharged early (≤ 6 days). The mean number of days of hospitalization in this group was 9.4 days (SD 17) and 2 patients (13%) required ICU admission. Three patients had a longer length of stay and were discharged after 6 days. Of these, two developed organizing pneumonia and one had bacterial pneumonia.

On the other hand, 14 cases (48%) had persistent sgRNA positivity after RDV treatment. The mean number of days of hospitalization in this group was 24 (SD 35). Four of these patients (29%) required ICU admission and the hospital stay was longer compared to the rest of the cohort. The three deaths recorded in the study were in this group.

In the subgroup of patients with positive sgRNA at baseline, 12 out of 15 patients (80%) were discharged early (≤ 6 days) with negative sgRNA at day 5 compared to 6 out of 14 patients (43%) with positive sgRNA at day 5 (p = 0.06).

Immunosuppressive treatment was maintained in patients presenting mild SARS-CoV-2 infection, whereas mycophenolate was discontinued in those with moderate or severe disease. Previous immunosuppressive therapies were also ceased while dexamethasone was administered, usually in patients presenting severe disease.

Table 3 shows the univariate and multivariate analyses of the factors associated with positive sgRNA after RDV treatment. The variables included were diabetes mellitus, high blood pressure, ischemic cardiopathy, prior transplantation, lymphopenia, previous hospitalization, vaccination with 3 or more doses of mRNA SARS-CoV-2, high dose of mycophenolic acid (≥ 1500 mg/day), mammalian target of rapamycin regimen, previous SARS-CoV-2 infection, acute allograft rejection (6 months previously), infection in the first year after transplantation, severe illness (ICU admission), treatment with dexamethasone or monoclonal antibodies, median quantitative gRNA at admission, readmission due to SARS-CoV-2 infection, and death (30 days). In the univariate analysis, age, the presence of diabetes mellitus, and quantitative gRNA at baseline were more frequent in the group of patients in whom sgRNA remained positive after RDV treatment, almost reaching statistical significance. Nevertheless, no association could be established in the multivariate analysis.

Table 3 Univariable and multivariable analysis on factors associated with positive SgRNA post remdesivir treatment in the population with a positive sgRNA at admission
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Discussion

In our study, we found a clinical correlation with qualitative sgRNA positivity. Importantly, we think that sgRNA could be of possible greater clinical utility for monitoring virological response to RDV compared to gRNA in KTR with SARS-CoV-2 infection. To our knowledge, this is the first study analyzing the utility of sgRNA in SARS-CoV-2 infected solid-organ transplant recipients.

A previous study including 117 patients treated receiving RDV described sgRNA as a good potential prognostic and monitoring marker, with multivariate analysis showing that the variables associated with early discharge and survival were negative sgRNA on day 3 and no need for dexamethasone concomitant treatment or ICU admission [17].

The results of our study showed that 14.7% (5 of 34) of KTR with documented SARS-CoV-2 infection were sgRNA negative in the initial respiratory sample. They presented mild respiratory symptoms, the median number of days from symptom onset to NPS was > 7 days, and none died. All these patients remained sgRNA-negative after RDV treatment (except one patient who switched to positive within 24 h of the first test). Therefore, the need for antiviral treatment in this subgroup of patients is open to discussion for several reasons. First, the efficacy of RDV when administered early (≤ 7–10 days from symptom onset) has been demonstrated, and, second, a sgRNA-negative result is probably indicative of non-viable virus. In line with this, in a previous study by our group including 117 patients who received RDV, we found that there was also a relationship between negative sgRNA and early discharge [17]. Nevertheless, more data are needed to confirm our results.

Of the 15 patients with positive sgRNA and a subsequent negative sgRNA after RDV treatment, 12 (80%) were discharged early. No patient in this group required ICU admission or died. Three patients in this group had a longer hospital stay and were discharged after 6 days due to other complications. We interpreted that, in this subgroup of patients, the antiviral treatment was effective and the absence of virus viability translates into a good clinical outcome.

Finally, the last subgroup of patients was sgRNA-positive both before and after RDV treatment. These patients did not clear sgRNA and had a longer length of hospital stay. Alonso et al. also described that positive sgRNA after antiviral treatment was associated with worse outcomes. All the patients who died in our study were included in this group. These results are in line with previous studies showing that mortality is associated with viral load at admission [9].

There is emerging evidence showing that prolonged viral shedding occurs in immunosuppressed patients, such as solid-organ transplant recipients, especially those receiving lymphocyte-depleting antibodies, monoclonal antibodies, such as rituximab, or high doses of steroids [18]. Recent studies that included KTR found that the use of triple combination therapy with two antivirals and monoclonal antibodies was associated with a high rate of virological clearance and clinical response [19]. In these cases, sgRNA could be helpful in defining candidates requiring more aggressive antiviral therapy.

The role of sgRNA as a surrogate marker of viral viability is promising [13, 20, 21]. In fact available studies have shown that sgRNA becomes undetectable earlier than gRNA, and, therefore, sgRNA determination may be of great value for clinical decision-making, such as when to initiate and discontinue antiviral therapy. Indeed, a recent study by our group [17] consistently supported the efficacy of sgRNA as a surrogate marker of infectivity.

The major limitation of the present study is that the number of patients included was small, as these are complex patients with specific characteristics. It would be necessary to continue the study prospectively. Secondly, this was a single-arm study and there was no control without antiviral treatment or other antivirals, as RDV was the standard of care in our institution, and it would have been unethical not to treat these patients. Finally, we could not compare these techniques with viral cultures, which is currently the gold standard technique.

Conclusions

We found that patients presenting negative sgRNA values at baseline presented better outcomes and, therefore, may not require antiviral treatment. Furthermore, a negative sgRNA result at day 5 was common in our KTR and correlated with a shorter hospital stay, supporting the duration of this therapeutic regimen. On the other hand, persistence of sgRNA at day 5 was associated with a worse outcome in our cohort of KTR. In this scenario, the prolongation of antiviral therapy or the combination of different antiviral therapies may be beneficial, although further prospective studies are needed.