*4.9. COVID-19 Convalescent Plasma (CCP)*

One of our patients in this case series was treated with convalescent plasma collected from recovered COVID-19 patients (CCP). Currently, add-on CCP, in addition to remdesivir and dexamethasone, can be considered in patients with an immune-deficient state, such as after SOT receiving immunosuppressive therapy, in HIV/AIDS, after aplasia-inducing chemotherapy before neutrophils recovery. This is a passive immune therapy, that currently being evaluated in clinical trials. It has been shown that most individuals with laboratorydiagnosed SARS-CoV-2 infection develop not only measurable antibody responses, but

also neutralizing antibodies [45]. The neutralizing antibody levels decline within the first 3 months following diagnosis, which suggests the collection of convalescent plasma with high neutralizing antibody concentrations may be optimally performed within a short time window after the infection has resolved [45]. Studies on CCP suggest improved clinical outcomes including radiological resolution, reduction in viral loads and improved survival. Most data relating to CCP treatment comes from the non-transplant population. The study of Duan et al. showed rapidly increasing neutralizing antibodies, and significantly improved clinical symptoms along with an increase of the oxyhemoglobin saturation within 3 days. In addition, improvements of lymphocyte counts, decreased C-reactive protein and various degrees of resolution of lung lesions in the radiological examinations were observed [46].

Moreover, there was no evidence of clinical hyperimmune responses after CCP treatment in a case series with 20 critically ill patients and 20 controls [47]. In a large series of 5000 hospitalized adults with severe or life threatening COVID-19, with 66% requiring intensive care unit treatment, the transfusion of CCP showed that the mortality rate was not excessive, and suggested that transfusion of CCP is safe in hospitalized patients with COVID-19 [48]. In that series, the incidence of all serious adverse events (SAEs) in the first four hours after CCP was <1%, including a low mortality rate (0.3%) [48]. Among the SEAs were mortality (n = 4), transfusion-associated circulatory overload (TACO; n = 7), transfusion-related acute lung injury (TRALI; n = 11), and severe allergic transfusion reactions (n = 3) [48]. In an uncontrolled case series of five critically ill patients with COVID-19 and ARDS, CCP treatment showed clinical improvement, and in four patients, ARDS resolved at 12 days after CCP, and three patients were weaned from mechanical ventilation within 2 weeks of treatment [49]. Although this treatment is promising, data on LTRs with COVID-19 treated with CCP are still lacking. Our patient who received CCP survived COVID-19 and did not show clear adverse events from the CCP treatment. However, caution with CCP in LTRs is certainly advisable, since CCP in immunosuppressed patients has also been associated with the emergence of new SARS-CoV-2 variant populations in these patients. These viral mutants are more likely to arise in immunocompromised patients, as they have a higher viral burden, increasing the opportunity for variant selection [50]. Therefore, CCP use for COVID-19 in LTRs could give rise to SARS-CoV-2 mutations. This has been observed in immunosuppressed patients treated with CCP for COVID-19 [51].

This hypothesis was confirmed in animal experiments, showing that CCP resulted in antibody-resistant SARS-CoV-2 variants, including the E484K mutation associated with vaccine resistance [52]. A comparable mechanism with emergence of resistant variants has been observed in immunocompromised patients with influenza infections who received long-term oseltamivir treatment [53]. The emergence of SARS-CoV-2 variants may lead to infections in COVID-19 vaccinated LTRs or in those who have survived COVID-19.

#### *4.10. Respiratory Co-Infections*

In our study, 15 patients (83%) were treated with broad-spectrum antibiotics including co-amoxicillin, tazobactam-piperacillin and meropenem, in order to prevent or treat respiratory co-infections. In all 18 patients, we found no evidence of respiratory co-infections, although these infections cannot be completely ruled out.

Estimations of the prevalence of co-infections among COVID-19 patients range from 0% to 45% [54]. Most of the co-infections occur within the first 4 days after infection55, more commonly in SOT recipients [55,56].

In SOT recipients with COVID-19, respiratory secondary co-infections have been addressed in a large number of studies, showing bacterial, viral and fungal secondary infections [57]. Bacterial secondary co-infections were due to Gram negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanni, Klebsiella pneumoniae, Morganella morganii, and Stenotrophomonas maltophilia, as well as Gram positive bacteria, including Enterococcus faecalis, Staphylococcus aureus, and Streptococcus haemolyticus [57].

Several authors reported viral secondary co-infections due to Cytomegalovirus (CMV) infection [58]. Although a secondary co-infection with influenza virus was demonstrated in a liver transplant recipient, this has not been described in LTR yet [59]. Aspergillus fumigatus [60] and Aspergillus niger [61] are also known to cause secondary co-infection in LTR.

While bronchoscopy is not available or advisable in routine care of COVID-19 patients, it is an option in mechanically ventilated ICU patients, providing microbiological samples from broncho-alveolar lavage or bronchial wash samples. Alternatively, samples can be obtained from (blind) tracheal aspirates.

## *4.11. Immunosuppressive Therapy during COVID-19*

In LTRs affected by COVID-19, the optimal management of immunosuppression still remains an unanswered question. Our patients in this case series were all on long-term immunosuppressive therapy. Immunosuppression causes lymphopenia, being a risk factor for severe COVID-19. Moreover, mycophenolate as well as mTOR inhibitors, can impair the immune response to viral (and bacterial) infections. Therefore, in COVID-19, mycophenolate often will be reduced or discontinued, as we also did during the SARS-CoV-2 infection. The risk of decreasing or pausing mycophenolate should be weighed against the risk of transplant rejection. This is in line with the current recommendation of the International Society of Heart and Lung Transplantation (ISHLT), advising to hold mycophenolate mofetil, mTOR inhibitors or azathioprine in the context of hospital admission with moderate/severe COVID-19 [7].

On the other hand, calcineurin inhibitors (CNIs) may prevent or attenuate the cytokine storm, by inhibiting interleukin (IL)-6 and IL-1 pathways, and therefore are maintained in most patients. In our 18 patients, we did not reduce or discontinue CNI.
