key: cord-1026931-1ysn0kyr
authors: Christensen, Johanna; Kumar, Dhiren; Moinuddin, Irfan; Bryson, Alexandra; Kashi, Zahra; Kimball, Pamela; Levy, Marlon; Kamal, Layla; King, Anne; Gupta, Gaurav
title: Covid-19 Viremia, Serologies and Clinical Course in a Case Series of Transplant Recipients
date: 2020-09-03
journal: Transplant Proc
DOI: 10.1016/j.transproceed.2020.08.042
sha: ece5c7d419fa066ca1417b162295165a38fb4e5e
doc_id: 1026931
cord_uid: 1ysn0kyr

Here we report a single-center cohort of 6 patients (4 kidney-only, and 2 simultaneous liver/kidney transplants) diagnosed with COVID-19 at a median of 1.9 years (range=0.2-9.3 years) post-transplant. Five (of 6) patients required inpatient admission, two patients (mortality=33%) died. Among those with mortality, an increased concentration of inflammatory biomarkers [interleukin-6 (IL6) and C-reactive protein] was noted with a lack of response to IL-6 blockade, remdesivir and/or convalescent plasma. None of the kidney-only transplants (4/6; 67%) had elevation in plasma donor-derived cell-free DNA above the previously published cut-off of 1% suggesting absence of significant allo-immune injury. Four (of 5) admitted patients had detectable SARS-CoV-2 (severe acute respiratory syndrome–coronavirus 2) in blood on samples obtained at/during hospitalization. Of the 4 discharged patients, two patients with undetectable virus on repeat nasopharyngeal swabs had seroconversion with positive SARS-CoV-2 IgG formation at 30-48 days post-infection. One patient had prolonged shedding of virus on nasopharyngeal swab at 28 days post-discharge despite lack of symptoms. In this preliminary report, we find that immunocompromised transplant patients had higher rates of RNAemia (67%) than reported in the general population (15%), seeming absence of allo-immune injury despite systemic inflammation and formation of IgG overtime after recovery from infection.

A number of case series have described the clinical manifestations and high mortality (15-30%) of COVID-19 in KT recipients. [3] [4] [5] There has been a significant decline in kidney transplants all over the world regardless of prevalence of the disease. 6 Studies describing SARS-CoV-2 viremia, serological responses in immunosuppressed patients and response to modulation of immunosuppression are the needed next step. Several unknowns remain. First, it is unclear as to which factors determine the severity of clinical course of disease in immunocompromised patients. Secondly, it is unknown whether COVID-19 triggers allo-immunity and rejection. It is plausible that the cytokine release syndrome associated with COVID-19 and reduction in immunosuppression may predispose KT patients to rejection. 7 Thirdly, while studies in the general population suggest rapid formation of IgG within a few days of COVID-19 infection; this data is unavailable for immunocompromised KT patients who have a blunted response to both viral infections and vaccines. 8, 9 J o u r n a l P r e -p r o o f In this first case series, we report the characteristics, inflammatory immune response, biomarkers of graft injury along with SARS-CoV-2 RNAemia and serological response in a small cohort of kidney/liver transplant patients.

Patient Selection:

Between, March 2020 and May 2020, six symptomatic kidney transplant recipients presented to the Virginia Commonwealth University hospital and tested positive for SARS-CoV-2. These adult (age >18 years) solid organ transplant (SOT) recipients were retrospectively assessed.

All initial tests to diagnose COVID-19 used qualitative real-time reverse transcriptase PCR (RT-PCR) of nasopharyngeal (NP) swab specimens. The study was approved by the VCU Institutional Review Board.

A commercial real-time fluorescent RT-PCR kit (BGI Genomics Co. Ltd, Shenzhen, China) was used to detect RNAemia from plasma samples collected at the time of diagnosis. The kit contains a sequence-specific fluorescent probe that uses FAM as a reporter and an internal reference probe that uses VIC/HEX as a reporter. The total reaction volume was 30 µL and was set-up according to the manufacturer's protocol. The reaction procedure was 50°C for 20 minutes, 95°C for 10 minutes, 

Venous blood was collected in Streck Cell-Free DNA blood collection tubes and shipped to the central Clinical Laboratories Improvements Act certified laboratory at CareDx, Inc. Details of the standardized specimen processing and analytical methods to determine the percentage of dd-cfDNA (AlloSure®) have been published. 10 The targeted next-generation sequencing assay employs highly polymorphic single nucleotide polymorphisms to quantify dd-cfDNA without need for separate genotyping of the recipient or the donor.

Between March 2020 and May 2020, four kidney-only transplant (KT) recipients (67%) and two simultaneous liver kidney transplant (SLKT; 33%) recipient tested positive for COVID-19 PCR by NP swab specimen. The demographics of these patients are presented in Table 1 . The median age was 56 years (range: 51-69). Four of six (67%) were African American and majority of patients had a history of hypertension (6/6; 100%) and diabetes (5/6; 83%). The median time from transplant was 1.9 years (range: 0.21-9.3 years).

All patients presented with the triad of fever, dyspnea and cough. They were negative for any other viral or bacterial respiratory infections. Only two (33%) patients required supplemental oxygen (2-4L/min) at the time of presentation. Four (67%) patients had infiltrates on chest imaging. Three (50%) patients had diarrhea at the time of presentation. Sequential Organ Failure Assessment (SOFA) score on average at the time of presentation was 2±1.6. Only one patient presented with acute kidney injury (AKI) related to prerenal causes from diarrhea for several days prior to presentation. There were no cases of new onset proteinuria or active urine sediment.

Laboratory biomarkers are presented in Table 2 . At presentation none of the patients had leukopenia with a mean absolute neutrophil count (ANC) of 4600±1982 per mm 3 . Majority of the patients (4/6, 67%) were profoundly lymphopenic (lymphocyte count < 1000 per mm 3 ) and the mean absolute J o u r n a l P r e -p r o o f lymphocyte count was 717±474 per mm 3 .

One patient with fever, cough and dyspnea but no infiltrate was discharged with reduction of Mycophenolate mofetil (MMF) dose by 50% (Patient 1). He was seen weekly by telemedicine and improved. Those that remained out of ICU were discharged in improved condition (3/6; 50%; Patients 2, 5 and 6). The two patients (33%: Patients 3 and 4) who developed hypoxemic respiratory failure required mechanical ventilator, vasopressor and renal replacement support. Eventually both these patients died. Of the two deaths one was the recipient of SLKT, and the other was KT only recipient with a history of chronic antibody mediated rejection.

All interventions are summarized in Table 3 . All patients (83%) admitted to the hospital had their MMF stopped at the time of admission. The only patient managed as an outpatient had MMF reduced by 50%. Two (33%) received remdesivir for treatment, one of whom was discharged in stable condition. Both the patients who were in the ICU, received IL6 blockade and one of those received convalescent plasma.

All KT only recipients had low level donor derived cell free DNA (dd-cfDNA) at the time of presentation (<1%). Initial testing for dd-cfDNA was done prior to adjustment of immunosuppression. The mean dd-cfDNA in the KT only recipients J o u r n a l P r e -p r o o f was 0.32±0.06%. Two patients who underwent subsequent dd-cfDNA testing on reduced immunosuppression within two weeks did not show any clinically significant change (0.26% to 0.33% and 0.41% to 0.39%). In SLKT recipients the dd-cfDNA is not validated however our main purpose was to establish a baseline which we could follow in order to guide future immunosuppressive management while correlating with allograft function and clinical response to infection. As expected, both these patients had higher average dd-cfDNA of 5.6±0.8% as compared to their KT only counterparts. They did not show any evidence of biochemical liver dysfunction. Four weeks later while on reduced immunosuppression the surviving SLKT recipient (Patient 2) had a decline in dd-cfDNA from 4.8% to 1.2%.

At presentation the markers of inflammatory response specifically interleukin-6 (IL6) (mean: 104±109 pg/ml), C-reactive protein (CRP) (Mean: 10.6±8.56 mg/dl) and ferritin (mean: 851±782 ng/ml), were high. The two patients who presented with the highest IL-6 and CRP levels (Patients 3 and 4; Table 2 ) showed rapid progression of disease and eventually died. 

The cycle threshold (Ct) for the qualitative RT-PCR testing for the NP swab was further evaluated (Table 2) 

There are significant knowledge gaps with regards to COVID-19 in KT patients. In this small case series, we confirm several findings from previous publications:

higher mortality compared to the general population (2/6; 33%), presence of comorbidities, co-existence of lymphopenia and the association of inflammatory markers with outcomes. In addition, we present novel findings.

In our case series we found that 80% (4/5) of patients who were tested for SARS-CoV-2 RNA in plasma tested positive for presence of circulating virus. Three of these patients tested positive at the time of presentation while the fourth tested positive on re-testing two weeks later. This may be due to a false negative initial RT-PCR that returned positive on re-testing. One patient without RNAemia at the time of presentation had evidence of neutralizing antibodies. This patient presented with a longer duration of symptoms, did not undergo lymphocyte depletion induction at the time of transplant and had been maintained on lower baseline immunosuppression. All factors that could have contributed to the lack of RNAemia at the time of presentation.

To date there have been no studies demonstrating RNAemia in SOT recipients.

However, this seems to be a much higher rate of RNAemia when compared to the few studies in the general population where RNAemia rates have been found to be ~15%. 11, 12 In fact, in one of our patients with severe disease, detectable virus was J o u r n a l P r e -p r o o f present even 2 weeks after presentation. Given the lack of host immunity, this data seems to raise the tantalizing possibility that the presence of SARS-CoV-2 RNA in blood may correlate with clinical diagnosis, hematogenous spread, extra-pulmonary manifestations and severity of disease. In our evaluation of the Ct values for the NP swabs and blood we found that those with most severe manifestations and death had lower Ct values indicating higher viral particles as compared to those who survived and improved. While ours was a qualitative assessment of RNA in the blood, in the future it will be interesting to further delineate if RNAemia is common and if the quantitative viral load may be of further informative value. This is particularly relevant as NP swabs are both subject to frequent false negatives and pose significant discomfort to patients. 13 One of our patients had evidence of viral shedding even after 28 days despite presence of SARS-Cov-2 IgG. Similar data among KT patients has been reported by other authors. 14 It is unclear whether this is infectious virus and further studies will be needed to further investigate this given implications of this for return to work, clinic environments and adjustment of immunosuppression.

We also report data on seroconversion in KTs for the first time. Both of our patients who recovered and were at least a month post-infection developed SARS-Cov-2 IgG. Both these patients were maintained on reduced dose MMF till the time of sero-conversion after which MMF was increased to the pre-infection dose with no short-term ill-effects. This is encouraging as it indicates that an impaired immunity may not prevent antibody formation. While it is not yet established if J o u r n a l P r e -p r o o f seroconversion confers immunity in the general population, 8, 9 the low re-infection rates and early reports of favorable efficacy of convalescent plasma in patients with severe COVID-19 manifestations [15] [16] [17] [18] suggest that this may be true.

Finally, we present data on markers of graft injury. AKI has been reported widely in COVID-19 infected immunocompetent patients. In an interesting autopsy report endothelialitis was reported in a transplant kidney. 19 It is unclear whether this represented allo-immune rejection or thrombotic microangiopathy related to COVID-19. While we did not see any evidence of systemic thromboembolism, autopsies were not performed on the two deceased patients. We do report that despite a robust immunological response to the virus as measured by inflammatory markers, we found that graft function and dd-cfDNA, a validated biomarker to detect immunological graft injury remained low at presentation allowing for reduction of immunosuppression. Given the almost universal presence of lymphopenia, elimination of MMF was necessary. In the two patients where consecutive dd-cfDNA results were available, we did not observe any significant elevation in dd-cfDNA despite a reduction in immunosuppression. Although the two SLK patients had higher average dd-cfDNA of 5.6±0.8% as compared to their KT counterparts, they did not have any evidence of liver dysfunction. The dd-cfDNA results on the SLK patients remained within the normal range based on the few studies on liver transplants and dd-cfDNA. 20, 21 Future larger studies will be required to further establish objective criteria to guide reduction and re-escalation of immunosuppression in KT patients with COVID-19 infections. ; IL-6= Interleukin-6 (0-12;pg/mL); CRP= C-reactive protein (0-0.5;mg/dL); Ferritin (16-150;ng/mL); N/A=Not available; NaB=SARS-CoV-2 Neutralization Antibody for all Ig isotypes; IgG= Immunoglobulin G; NP= Nasopharyngeal; dd-cfDNA= donor derived cell free DNA

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Similarity in Case Fatality Rates (CFR) of COVID-19/SARS-COV-2 in Italy and China

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Confirmed Cases in Wuhan, China

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Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury

Graft-derived cell-free DNA as an early organ integrity biomarker after transplantation of a marginal HELLP syndrome donor liver

Johanna Christensen: https://orcid.org/0000-0003-2389-1320