Increased Risk of Invasive Aspergillosis in Immunocompromised Patients With Persistent SARS-CoV-2 Viral Shedding >8 Weeks, Retrospective Case-control Study

Abstract Background Immunocompromised patients now represent the population most at risk for severe coronavirus disease 2019. Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral shedding was reported in these patients ranging from several weeks up to 9 months. We conducted a bicentric retrospective case-control study to identify risk and prognostic factors associated with persistent viral shedding in immunocompromised patients. Material and Methods Symptomatic immunocompromised adults with persistent SARS-CoV-2 viral shedding >8 weeks were retrospectively included between 1 March 2020 and 24 April 2022 at 2 university hospitals in Paris, France, and matched with a control group consisting of symptomatic immunocompromised patients without persistent viral shedding. Results Twenty-nine immunocompromised patients with persistent viral shedding were compared with 40 controls. In multivariate analysis, fever and lymphocytopenia (<0.5 G/L) were associated with an increased risk of persistent viral shedding (odds ratio [OR]: 3.3; 95% confidence interval [CI], 1.01–11.09) P = .048 and OR: 4.3; 95% CI, 1.2–14.7; P = .019, respectively). Unvaccinated patients had a 6-fold increased risk of persistent viral shedding (OR, 6.6; 95% CI, 1.7–25.1; P = .006). Patients with persistent viral shedding were at risk of hospitalization (OR: 4.8; 95 CI, 1.5–15.6; P = .008), invasive aspergillosis (OR: 10.17; 95 CI, 1.15–89.8; P = .037) and death (log-rank test <0.01). Conclusions Vaccine coverage was protective against SARS-CoV-2 persistent viral shedding in immunocompromised patients. This new group of immunocompromised patients with SARS-CoV-2 persistent viral shedding is at risk of developing invasive aspergillosis and death and should therefore be systematically screened for this fungal infection for as long as the viral shedding persists.

Three years after the start of the coronavirus disease 2019 (COVID-19) pandemic, in December 2023, the World Health Organization reported 772 million and 6.9 million cumulative cases and death, respectively [1,2].Immunocompromised (IC) patients now compose the population the most at risk for severe COVID-19 (intensive care unit [ICU], hospitalization, and death) [3][4][5][6].It represents at least 2%-3% of the US and EU population and includes patients with solid cancers and hematological malignancies, hematopoietic stem cell transplantation, solid organ transplant (SOT), inborn error of immunity, human immunodeficiency virus (HIV) (CD4 + T-cell count <200/mm 3 ), autoimmune diseases, and patients under immunosuppressives Increased Risk of Invasive Aspergillosis in Immunocompromised Patients • OFID • 1 Open Forum Infectious Diseases M A J O R A R T I C L E therapies [7][8][9].Because IC patients lack effective vaccine immune responses, whether humoral (in patients receiving B-cell-targeting therapies) or both humoral and cellular in SOT, a COVID-19 booster is recommended every 6 months to increase vaccinal response [7][8][9][10].Nonetheless, it remains a heterogeneous group of patients, in which the immunological vaccine response is largely uncertain, varying from 1 patient to another [8].Monoclonal antibody prophylaxis has therefore been an effective alternative for patients who do not develop postvaccinal immune responses, with encouraging results but that are limited by the emergence of new resistant variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [11,12].
Since the beginning of the pandemic, persistent viral shedding has been reported in IC patients varying from several weeks up to 9 months and new challenges have been raised [13][14][15][16].First, persistent contagiousness prompted the authorities to extend eviction until 20 days in these patients [17].Second, they were subject to prolonged hospitalization, late deterioration, and complications sometimes favored by the intensification of the immunosuppressive drugs to treat the underlying disease(s).Finally, persistent viral shedding in this group of patients raised major therapeutic concerns because antiviral drugs could not eradicate the virus and were responsible for the emergence of mutations, resistance, and new variants [13,[18][19][20].Several risk factors for persistent viral shedding had been identified, among which are B-cell dysfunction (malignant lymphoma, multiple myeloma, anti-CD20 and anti-CD19 therapies), lymphocytopenia, serum elevation of type 1 interferon, immature neutrophils, delayed antiviral therapy, and D-dimer elevation [15,16,[21][22][23].Persistent viral shedding was associated with a spike protein mutation and an increased risk of bacterial infections and death [15,16,[21][22][23].However, the absence of a standardized definition of the duration of "persistence" makes it difficult to interpret and extrapolate previous reports [16].
We report here a bicentric retrospective case-control study to identify risk factors for persistent viral shedding and prognostic factors among IC adults.

Study Design
We conducted a retrospective bicentric case control study including IC patients with persistent SARS-CoV-2 viral shedding >8 weeks.Patients were included from 2 different university hospitals, Cochin Hospital and Necker Enfants-Malades Hospital ("Paris-Centre hospital group"), and IC patients were included from the departments of infectious diseases and tropical medicine, nephrology, kidney transplantation, hematology, ICU, and internal medicine.The study was approved by the French Assistance Publique des Hôpitaux de Paris Research Ethics Committee "Comité d'éthique de la recherche APHP centre" (institutional review board registration: #00011928), in accordance with current standards.
The management of patients with COVID-19 within the "Paris-Centre" hospital group was comparable and homogeneous in all the departments considered for this study.It followed the recommendations of the multidisciplinary consultation meetings held monthly within the hospital group during the pandemic period and chaired by one of us (L.M.).

Definition of Patients
Persistent viral shedding was defined by the presence of repeated (≥2) positive SARS-CoV-2 polymerase chain reaction (PCR) tests on a nasopharyngeal sample for at least a period > 8 weeks without a negative nasopharyngeal SARS-CoV-2 PCR during this period.All patients with persistent viral shedding from 1 March 2020 to 24 April 2022 were included.Data collection included clinical and biological presentations at diagnosis, SARS-CoV-2 genotype, current treatment, specific anti-COVID-19 medication, and potential complications, including documented bacterial and fungal infections and outcomes.Clinical complications such as COVID-19-related invasive aspergillosis and mucormycosis were identified among the included patients according to the current guidelines [24,25].Probable and proven invasive aspergillosis, regardless of the site of infection (pulmonary and/or other), in patients who were not admitted in ICU were considered according to the European Organization for Research and Treatment of Cancer guidelines [26,27].Messenger RNA vaccines (Pfizer and Moderna available in France) and monoclonal antibodies prophylaxis (sotrovimab, casirivimab/imdevimab, tixagevimab/cilgavimab) were considered when administered before COVID-19 infection.A centralized second computed tomography (CT) scan imaging reading was performed in all patients by a single qualified radiologist (S.B.).We included IC patients with primary immune deficiency/inborn errors immunity, HIV infection with CD4-T cells < 200/mm 3 , autoimmune diseases, SOT, allogeneic hematopoietic stem cell transplantation, chronic lymphoid malignancies (treated or untreated) (ie, chronic lymphocytic leukemia, non-Hodgkin lymphoma and myeloma), and patients receiving immunosuppressive therapies such as chimeric antigen receptor-T-cell therapy or therapeutic bispecific antibodies, anti-CD20 antibodies or Bruton tyrosine kinase inhibitors, azathioprine, cyclophosphamide, methotrexate, mycophenolate mofetil, calcineurin inhibitors, corticosteroids, and belatacept.
Controls (n = 40) were selected as follows: (1) between 1 March 2020 and 24 April 2022 (during the same period as the cases); (2) were immunocompromised patients corresponding to the criteria defined previously; (3) were immunocompromised patients with symptomatic COVID-19 infection; (4) had proven positive SARS-CoV-2 PCR on nasopharyngeal specimen; (5) and proven negative for SARS-CoV-2 on PCR via nasopharyngeal specimen within ≤8 weeks after the first positive test; and (6) were followed up > 6 months in our centers with regular visits because of their immunocompromised status.
We excluded asymptomatic patients, patients for whom clinical data were unavailable, patients with reinfection (defined as negative PCR between 2 positive results or identification of 2 different SARS-CoV-2 variants), and patients who did not had a 6-month follow-up in our centers.

Statistical Analysis
To compare the distribution of continuous and dichotomous variables between 2 groups, we used the χ 2 or the 2-sided Fisher exact tests, respectively.All tests were 2-sided, and P < .05 was considered significant.Indicative factors for persistent viral shedding, death, and invasive aspergillosis (IA) were determined using the logistic regression model.The Kaplan-Meier method was used to determine cumulative probabilities of all-cause death and cumulative probabilities of IA, with between-group comparisons of cumulative event rates calculated using the log-rank test.All analyses were performed using SPSS version 29.0 statistical software.

RESULTS
During the study period, 1982 patients had a positive SARS-CoV-2 PCR result on nasopharyngeal samples at Cochin Hospital-University and 521 at Necker Enfants-Malades Hospital-University.Of these, 266 had at least 2 positive SARS-CoV-2 PCRs on nasopharyngeal samples over 8 weeks.A total of 237 were excluded because of reinfection (ie, negative PCR between 2 samples or identification of 2 different variants), because they were asymptomatic (n = 6), non-IC patients (n = 21), or because of unavailable data (n = 48).Overall, the incidence of persistent viral shedding > 8 weeks from those 2 centers was 1.4% (Figure 1).

Characteristics of Patients With Persistent Viral Shedding (>8 Weeks)
We identified 29 patients with persistent viral shedding.The mean age was 57.7 ± 16 years and 55.2% were men.Most patients, 55.2% (n = 16) were SOT recipients, including 13 kidney transplant recipients (KTRs), 1 kidney and liver transplant recipient, and 2 lung transplant recipients.Ten patients presented hematological diseases (Table 1; Supplementary Figure 1).Two patients had concomitant SOT and hematological disease, including 1 KTR with a diffuse and large B-cell lymphoma and 1 lung transplant recipient with acute myeloid leukemia.One patient with HIV developed lymphoma and 4 had other immunodeficiencies (Table 1; Supplementary Figure 1).Ninety-three percent of these patients (n = 27) were receiving immunosuppressive drugs at the time of diagnosis and 46% (n = 22) had received long-term corticosteroid   1 and Supplementary Figure 1.

Complications, Invasive Aspergillosis and Death
Among the 29 patients with persistent viral shedding, 6 (20%) presented IA compared with 1 in the control group.The mean duration of SARS-CoV-2 viral shedding in these patients was 142 ± 137 days.The delay between first positive SARS-CoV-2 PCR and IA was 100 ± 133 days.All of them had invasive pulmonary aspergillosis.One patient developed proven chondrosternal IA (he received pulse methylprednisolone in the ICU and had a concomitant probable pulmonary aspergillosis and Klebsiella pneumoniae pneumonia).One patient had both IA (Aspergillus fumigatus) and severe mucormycosis (Rhizopus arrhizus) (Supplementary Figure 3).All of these patients received azole therapy and 2 died, 81 and 125 days after IA diagnosis (Table 2).Eight patients with SARS-CoV-2 persistent viral shedding died.Five had hematological malignancies, 2 were KTRs, and 1 had a combined immunodeficiency (Table 2).Five were hospitalized in the ICU: 4 of them presented documented Pseudomonas aeruginosa pneumonia and 3 had IA (2 probable and 1 possible).For 7 of these patients, death was related to complications of COVID-19, bacterial superinfections, or AI, and 1 patient died from progression of his hematological malignancy (Table 3).

DISCUSSION
In the present study, we identified an emerging group of patients with dramatic persistent SARS-CoV-2 viral shedding who presented an approximate 5-fold increased risk of hospitalization, a 10-fold increased risk for IA, and a significantly increased risk for death, highlighting the potential disease severity in IC patients.
We report a large series of IC patients with persistent viral shedding, including 16 SOT patients (mostly KTRs).To the best of our knowledge, 88 IC patients with persistent viral shedding have been reported in 70 carefully analyzed articles in the literature.Most had hematological malignancies (n = 65) and a few other conditions (Supplementary Figure 5; Supplementary Table 2).According to these 88 cases, persistent viral shedding was reported with a mean viral shedding that was slightly longer than what we observed in our cohort.Sixteen of the patients reported in the literature died (18%) and 8 (9%) had IA, which is less than what we observed here (27% and 20.6%, respectively) [13,[28][29][30][31][32][33][34].Recently, virus-associated pulmonary aspergillosis (influenzae-and COVID-19-associated pulmonary aspergillosis) confirmed the invasiveness of aspergillosis in a series of autopsies carried out on patients who had been hospitalized in the ICU [26].Proven invasive pulmonary aspergillosis diagnosis presented a similar histological pattern in influenza and in COVID-19 ICU case fatalities, highlighting the need to consider this invasive fungal infections in patients with COVID-19 [26].Invasive aspergillosis can occur in the setting of viral infections because of virus-induced alteration of the respiratory epithelium and modification of immune responses, a phenomenon accentuated by mechanical ventilation and corticosteroid therapy in the ICU [35,36].In immunocompromised patients not hospitalized in the ICU, this mechanism is supported by the absence of cellular and humoral responses against both agents, virus and fungus [37].Nevertheless, the exact pathophysiology leading to invasive aspergillosis in non-ICU immunocompromised patients infected with SARS-CoV-2 has not been clearly identified.Several SARS-CoV-2 mutations in IC patients were described in the literature; some were involved in viral resistance to remdesivir, nirmatrelvir/ritonavir, and monoclonal antibodies, explaining the persistence of the viral shedding in those patients [18,20,[38][39][40][41][42][43].In a recent study of 150 IC patients with Omicron SARS-CoV-2 infection, only 9 patients had persistent viral shedding for more than 8 weeks [16].
According to the literature, we identified that vaccination was protective against persistent viral shedding in IC patients [44].Unvaccinated patients had a significant 6-fold increased risk  of persistent viral shedding.In the literature, 6%-69% of the SOT patients reached serological response with a third dose of vaccine.Thus, 24% of KTRs remained seronegative after a third dose of vaccine, and a lower neutralization immune response against evasive variants was observed in IC patients compared with immunocompetent patients after receiving a booster [45][46][47][48].Although implementing preventive measures (vaccination and monoclonal antibodies) may be insufficient in this group of patients, the therapeutic challenge has not yet been resolved.We observed that persistent viral shedding was significantly associated with plasma therapy and combined therapy (monoclonal antibodies and plasma therapy).Some authors reported the efficacy of a prolonged use of nirmatrelvir/ritonavir for more than 20 days in association with remdesivir for more than 19 days after failure of monoclonal antibodies, intravenous remdesivir, and prolonged oral corticosteroids, which were not used here [49].Recently, Mikulska et al proposed combination therapy with 2 antivirals and monoclonal antibodies in symptomatic IC patients with Omicron persistent viral shedding [44].They showed that the 14-and 30-day responses were significantly better when combination therapy included monoclonal antibodies [44].According to the literature review, most patients received combined therapies associating remdesivir, monoclonal antibodies, and convalescent plasma, without systematic viral clearance.To date, no standardized recommendations are available for IC patients with persistent viral shedding.
Our study has some limits.We likely underestimated the number of patients with persistent viral shedding because some patients had no control nasopharyngeal PCR, others died with a positive SARS-CoV-2 infection without control, and because we lacked clinical data for most of them.We did not observe a significant association between persistent viral shedding, B-cell depletion, and the use of anti-CD20 or CD19 antibodies, although it was reported in the literature [19,38].This could be linked to a lack of power resulting from the small number of patients suffering from hematological disorders and treated with B-cell-depleting therapies, as well as the high proportion of SOTs who did not receive anti-B therapies.
In conclusion, our study showed that vaccine coverage against SARS-CoV-2 was protective against persistent viral shedding in IC patients.Immunocompromised patients with persistent viral shedding were at risk for IA and death.This population should therefore be systematically screened for IA using galactomannan antigen and followed up with systematic CT scans to identify lung lesions suggestive of IA and perform bronchoalveolar lavage to confirm the diagnosis.

Table 1 . Characteristics of Patients in Persistent Viral Shedding and Control Groups (Univariate Analysis)
(n = 21) presented with an inflammatory syndrome (C-reactive protein >10 mg/L at the time of SARS-CoV-2 diagnosis).Characteristics of patients and underlying conditions are described in Table

Table 2 . Invasive Aspergillosis in Immunocompromised Patients With Persistent Viral Shedding
Increased Risk of Invasive Aspergillosis in Immunocompromised Patients • OFID • 7