Associations between invasive aspergillosis and cytomegalovirus in lung transplant recipients: a nationwide cohort study

Cytomegalovirus (CMV) and invasive aspergillosis (IA) cause morbidity among lung transplant recipients (LTXr). Early diagnosis and treatment could improve outcomes. We examined rates of CMV after IA and vice versa to assess whether screening for one infection is warranted after detecting the other. All Danish LTXr, 2010–2019, were followed for IA and CMV for 2 years after transplantation. IA was defined using ISHLT criteria. Adjusted incidence rate ratios (aIRR) were estimated by Poisson regression adjusted for time after transplantation. We included 295 LTXr, among whom CMV and IA were diagnosed in 128 (43%) and 48 (16%). The risk of CMV was high the first 3 months after IA, IR 98/100 person‐years of follow‐up (95% CI 47–206). The risk of IA was significantly increased in the first 3 months after CMV, aIRR 2.91 (95% CI 1.32–6.44). Numbers needed to screen to diagnose one case of CMV after IA, and one case of IA after CMV was approximately seven and eight, respectively. Systematic screening for CMV following diagnosis of IA, and vice versa, may improve timeliness of diagnosis and outcomes for LTXr.


INTRODUCTION
Lung transplanted recipients (LTXr) receive lifelong immunosuppressive treatment increasing the risk of opportunistic infections (1,2).Cytomegalovirus (CMV) and invasive aspergillosis (IA) account for a large burden of disease in LTXr, both being associated with an increased risk of graft rejection and high mortality rates (1)(2)(3).
About 28-68% of LTXr develop CMV infection (4) which requires treatment (5)(6)(7).Ganciclovir is often used to treat CMV but can cause neutropenia (8).The neutrophil granulocytes are a key element in the immune defense against fungal infections, and thus, a lack of neutrophils might increase the risk of fungal infections (9,10).Furthermore, CMV in itself can cause leukopenia (4) and is shown to have immunomodulating abilities which can increase the risk of infections (11).
Invasive aspergillosis is seen in 4-15% of LTXr, most frequently affecting the respiratory tract (12,13).Aspergillus has immunomodulatory properties through gliotoxin excretion (14) which may increase the risk of concomitant infection with CMV.IA is commonly treated with triazoles that interact with immunosuppressants, especially calcineurin inhibitors (CNI), and treatment with triazoles can cause increased plasma concentration of CNI leading to over-immunosuppression and increased risk of opportunistic infections, including CMV.
Cytomegalovirus is associated with a higher risk of IA in both solid organ transplant recipients (SOTr) (15)(16)(17) and hematopoietic stem cell transplant recipients (18).Earlier studies have shown an approximately 5-fold increased risk of invasive fungal infection in SOTr with CMV infection (19), but analyses were not adjusted for time after transplantation and detailed descriptions of this association, including absolute risks and the timing of these infections, are lacking.IA has the potential to increase the risk of CMV, as described, but this hypothesis has yet to be investigated in a clinical setting (19,20).
At the Copenhagen University Hospital, Rigshospitalet, an individualized program of CMV prophylaxis, monitoring and pre-emptive treatment has been implemented for all SOTr, based on CMV serostatus of donor and recipient (21).The program has reduced the number of CMV disease cases significantly in SOTr apart from LTXr where -no significant reduction in rates of CMV disease was seen (21).This indicates that the program could be tailored further for capturing CMV early in LTXr.Antifungal prophylaxis is complicated due to high rates of adverse events and drug-drug interactions associated with the commonly used triazoles (22).IA is difficult to diagnose and often requires bronchoscopy.There is currently no consensus for IA prevention, but targeted monitoring of patients at highest risk and pre-emptive therapy might be a beneficial approach (23).
We examined incidence rates of CMV after IA and vice versa in a clinical setting to assess whether screening for one of these infections may be indicated when the other is diagnosed.

MATERIALS AND METHODS
We performed a retrospective cohort study including all Danish patients 16 years of age or older, who received a lung transplantation (LTX) in the Danish National Lung Transplant Centre, Copenhagen University Hospital, Rigshospitalet, between 1.1.2010and 31.12.2019.All LTX in Denmark are performed at this centre.

Data collection
Data were obtained from the Personalized Medicine of Infectious Complications in Immune Deficiency (PERSI-MUNE) data warehouse, which contains nationwide data on transplantation, microbiology, pathology, and biochemistry (24).Furthermore, data on symptoms, bronchoscopy, and radiology findings were collected by review of medical charts and organized in a RedCap database (25).

Invasive aspergillosis
IA was defined in accordance with the ISHLT criteria (22).All positive mycological tests from LTXr were identified through automated search of the electronic national microbiological registry.A first round of medical chart review was performed, dividing cases into colonization or potential IA.All potential IA cases were then reviewed and classified independently by two expert reviewers.If the two reviewers disagreed on the classification, the case was evaluated by the entire expert review board, consisting of five medical doctors with clinical expertise in the field, where consensus was found.

Cytomegalovirus
CMV was defined as the detection of nucleic acid by polymerase chain reaction (PCR) in two consecutive plasma samples within 14 days of ≥300 IU/mL or one plasma or BAL sample with a viral load (VL) ≥3000 IU/mL (15).PCR analyses were performed as previously described (26).

Standard protocols
Standard protocols for prophylaxis were as previously described (21,22) and are summarized in supplemental material.The protocols for screening for CMV and IA are depicted in Fig. 1.

Statistical analyses
Patients were followed from the date of transplantation until 2 years after transplantation, death, emigration, retransplantation or 31.12.2020,whichever came first.The time from transplantation to IA and CMV was visualized by Kaplan-Meier graphs.We estimated incidence rates and incidence rate ratios using Poisson regression analyses.
Risk of IA and CMV was analyzed in two multivariable models with time-updated variables: Firstly, we analyzed risk of getting IA after CMV in a multivariable Poisson regression model.In the model, we included time after transplantation and CMV as time-updated variables, allowing us to compare the risk of IA in person-time after CMV to person-time before CMV, without confounding related to time after transplantation.Additionally, we added the following pre-defined potential confounders of IA for adjustment in the analysis: Sex, age, calendar time, and IA high-risk group (cystic fibrosis, sarcoidosis, and retransplantation).
Secondly, we analyzed the risk of getting CMV after IA in a similar manner with IA and time after transplantation as time-updated variables.In this model, we also adjusted for other pre-defined potential confounders of CMV by including the following variables: Sex, age, calendar time, and CMV high-risk group (CMV IgG serostatus: D+/RÀ).
The calendar time periods, used for adjustment, were defined according to the year of transplantation: 2010-2013, 2013-2016, and 2016-2019.
The number needed to screen (NNS) to detect one case of IA was calculated as the number of patients with CMV who were tested for IA within 3 months after CMV diagnosis divided by the number of patients diagnosed with IA within 3 months after CMV.The same method was used for the calculation of NNS to detect one case of CMV within 3 months of IA.
The study was approved by the Danish National Board of Health (3-3013-1060/1/ approved 19.March 2020) and the Danish Data Protection Agency (RH-2016-47; approved 16.January 2019).Ethics approval is not required for this type of study in Denmark.

RESULTS
We included 295 LTXr.The median age was 53 years (IQR 43-58), and 146 (48.5%) were female.A total of 76 (25.8%)LTXr were in the IA high-risk group (including patients with cystic fibrosis, sarcoidosis, and retransplantation; full list in supplementary) and 57 (19.3%)LTXr had high-risk CMV serostatus (D+/RÀ).Patient demographics and characteristics are summarized in Table 1.The total follow-up time was 347 person-years of follow-up (PYFU) for CMV and 449 PYFU for IA.Within the first 2 years after LTX, 63 (21%) LTXr died, at a median time of 297 days (IQR 132-449) after transplantation, and four received a retransplantation during the study period.
CMV after IA Among the 48 LTXr with IA, 47 (97.9%) had a CMV PCR performed at some point after IA diagnosis; of these, 47 (97.9%) were screened with plasma samples and 17 (36.2%)were screened with both BAL and plasma samples.Within 3 months of IA diagnosis, 46 (95.8%) had a plasma CMV PCR performed, of which 13 (28.3%)also had a CMV PCR in a BAL sample performed.
In the first 3 months following IA, there was a tendency toward an increased risk of CMV compared with the period prior to/without IA, aIRR 1.42 (95% CI 0.65-3.10).The numbers needed to screen to diagnose one case of CMV within the first 3 months following diagnosis IA was approximately seven.

IA after CMV
Among the 128 LTXr diagnosed with CMV, 120 (93.8%) were screened for IA at some point thereafter, of these 119 (93%) had culture of BAL performed and 72 (56.5%) had a galactomannan antigen test performed on BAL.Within 3 months of CMV diagnosis, 103 (80.4%) were screened for IA; of these, 102 (79.7%) had a culture of BAL performed and 27 (21.1%)also had a galactomannan antigen test performed on BAL.
Among the 128 LTXr diagnosed with CMV, 16 (12.5%)developed IA at some point after CMV (Fig. 3B).The median time from CMV to diagnosis of IA was 86.5 days (IQR 27-139.5).Within 3 months after CMV diagnosis, 13 (10.2%)LTXr developed IA, IR 30 per 100 PYFU (95% CI 15-60) (Table 3).Eight of the 13 patients (61.5%) had a BAL sample that was PCR positive during their CMV course prior to IA.In the first 3 months following CMV diagnosis, there was an increased risk of IA compared with the period prior to/without CMV, aIRR 2.91 (95% CI 1.32-6.44)(Table 3).The numbers needed to screen to diagnose one case

DISCUSSION
In this nationwide study, we evaluated rates of CMV and IA in 295 lung transplant recipients who were followed for 2 years after transplantation.We investigated the risk of contracting one infection after being diagnosed with the other.We found an increased risk of IA following CMV, and a tendency to increased risk of CMV following IA, but not as pronounced.The absolute rates of one of these infections were high in the first 3 months after diagnosis of the other, and numbers needed to screen to detect one case of IA after CMV and vice versa were 7-8.
The majority of CMV manifested as postprophylaxis infection with the highest incidence rates 3-6 months following transplantation.The hybrid prophylaxis strategy in our centre, consisting of 3 months of valganciclovir prophylaxis followed by a pre-emptive approach, has previously been evaluated (21).The intention of a pre-emptive strategy is to detect viremia early and start treatment before progression to disease.This strategy was found to be successful in preventing CMV disease in SOTr apart from LTXr (21), indicating a need for better guidance for early detection of CMV in this population.Cytomegalovirus viral load has been suggested as an appropriate surrogate marker for CMV disease (27).We found that the median viral load in patients with CMV following IA was ten times higher than in patients without preceding IA, indicating higher risk of CMV disease in these patients, and perhaps a somewhat accelerated course of CMV following IA or a delay in diagnosis

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CMV AND IA AFTER LUNG TRANSPLANTATION of CMV.This may be related to the challenges of ensuring early detection of CMV in patients with compartmentalized CMV replication in the lower respiratory tract.Plasma screening has been shown to be insufficient for early detection of CMV in these cases, where direct sampling for CMV by bronchoscopy is needed for timely detection of the infection.Bronchoscopy is less regularly performed, which can lead to diagnostic delay (28,29).We found similar crude incidence rates of CMV (approximately 100/100 PYFU) in the 3 months after IA and in the period 3-6 months after transplantation, which in our cohort is the post-CMVprophylaxis period.In our centre and others, this high CMV incidence rate triggers the use of preemptive strategy after ending CMV prophylaxis to prevent delayed-onset post-prophylaxis CMV disease.Our findings of equally high incidence rates of CMV in the three-month period succeeding IA diagnosis suggest a similar need of CMV screening following IA as in the post-prophylaxis period.
To our knowledge, IA has not been investigated as a risk factor for CMV in a clinical setting previously, but evidence of a potential casual association has been put forward in ex vivo studies.Aspergillus has intrinsic immunomodulating capacities through the secretion of mycotoxins (30).Gliotoxin is the most in-depth studied mycotoxin and several immunomodulatory functions have been described, including suppression of macrophages and T-cells (9,10,14,31).The interplay between Aspergillus and CMV has also been investigated in models, demonstrating synergistic immunosuppressive effects in co-infected cells that favor the two pathogens (32).
We found that IA was diagnosed in 16% of the entire cohort, and most frequently in the early period after transplantation, 0-6 months.The risk of IA was significantly higher in patients who previously had had CMV.This has been demonstrated before in both mixed SOTr cohorts (15,33) and in LTXr exclusively (16,(34)(35)(36)(37).Our risk estimate was lower than in some earlier reports, which was related to the adjustment of time after transplantation in our analysis.Few previous studies have included the time from transplantation in analyses of CMV and IA association.The majority of patients with CMV prior to IA had CMV detected in BAL, indicating infection in the respiratory tract.A non-intact barrier in the airways could be favorable for fungal invasion and thereby contribute to the increased risk of IA after CMV.These pathogeneses have previously been demonstrated in patients infected with influenza and COVID-19 that secondarily acquired IA with fungi entering through damaged epithelial cells in the lungs (38).Immunosuppressive effects of CMV have been shown in several models (32,39,40), and this too could dampen the host defense against fungi.Antiviral treatment-related neutropenia is also a possible factor that could contribute to the increased risk of IA in relation to CMV (8).
However, we found the risk of IA to be increased in both 0-3 months and >3 months after CMV and a similar pattern of a prolonged increased risk for CMV after IA.These findings could suggest the two infections being 'opportunistic partners', affecting patients with common risk factors of the two infections such as over-immunosuppression, structural or genetic predisposition (41,42).
Regardless of the underlying mechanisms of the association between the two infections, our results suggest that an increased awareness of one infection in patients diagnosed with the other could help in ensuring early diagnosis and treatment.
Timely diagnosis of CMV and IA is important for outcomes.CMV viral loads increase with delays in diagnosis (26), and a high CMV viral load is associated with significantly increased risk of CMV disease (4).A delay in treatment of IA is associated with increased risk of death (43).Both CMV (44,45) and IA (3,46,47) have been shown to be independently associated with chronic lung allograft rejection (CLAD), a major cause of mortality in LTXr (48).Invasive fungal infections following respiratory viral infections have recently been shown to be associated with increased risk of CLAD (49) and IA following CMV may also reinforce the association with CLAD, but this has yet to be investigated.
The clinical course following lung transplantation is affected by several interacting factors.The prophylaxis used for CMV and IA affect the risk and timing of the two infections, including their potential interplay.Our center uses a hybrid approach with 3-month universal prophylaxis followed by systematic monitoring and preemptive therapy for CMV for all LTXr, except those with CMV serostatus DÀ/RÀ.Other centers use longer duration of CMV prophylaxis, as suggested by some guidelines (50,51).It is not known whether the duration of CMV prophylaxis affects the rates of IA.This needs to be investigated in multicenter studies including centers that have different strategies for duration of CMV prophylaxis.If the association between CMV and increased risk of IA, observed in this study, reflects a causal relationship, longer CMV prophylaxis may reduce rates of IA.
The numbers needed to screen estimates are correlated with the screening protocol used and should be interpreted in that context.In our centre, routine screening included close and tailored CMV screening but IA screening through BAL culture was less frequently performed.Screening for CMV with PCR has a high sensitivity whereas IA diagnosis is difficult with rather low sensitivity of most diagnostic tests.ISHLT criteria of probable IA also require imaging or bronchoscopy.These factors put together might result in underestimation of IA.Furthermore, the screening protocol might introduce a difference in diagnostic delay.It is likely that CMV was generally detected earlier in the infectious course compared with IA.This may skew our results toward more frequent findings of CMV prior to IA, which could be reflected in the more pronounced increased risk of IA after CMV than the other way around.
Our cohort is relatively large compared with other studies in the field, and to our knowledge we are the first to present results of CMV and IA analyses where time from transplantation and time from infections are included.Nevertheless, due to the relatively small number of endpoints, our study has limited statistical power bringing some uncertainty to our findings.Due to this, we were also unable to include additional variables that could be associated to the risk of infections, for example, leukopenia or over-immunosuppression.
This together calls for validation in cohorts with larger sample size and preferably where different prophylaxis strategies are used.
A major strength of the study is the nationwide design with access to microbiological, pathological, and clinical data in electronic national registers, which can be linked by the Danish civil registration number register.Consistent screening protocols were used throughout the study period, and classification and validation of IA was performed in a systematic review process in a uniform manner for all patients with positive mycological findings.

CONCLUSION
In conclusion, we found increased risk of CMV after IA and vice versa.Enhanced screening after diagnosis of one of the infections may improve early diagnosis of the other and thereby potentially improve outcomes of LTXr.Validation studies in large multicenter cohorts are warranted.
We thanks the contributing patients and the MATCH team for enabling this study.

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2023 The Authors.APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.577 CMV AND IA AFTER LUNG TRANSPLANTATION of IA within the first three following diagnosis of CMV was approximately eight.

Fig. 2 .
Fig. 2. Kaplan-Meier graphs representing the probability of freedom from CMV (A) and invasive aspergillosis (B) over time after lung transplantation.

1
Adjusted for age > 50 years, sex, and IA high-risk underlying condition.2 Adjusted for time after transplantation, age > 50 years, sex, IA high-risk underlying condition, and calendar time.

Fig. 3 .
Fig. 3. Time of CMV and IA diagnosis in patients infected with both after lung transplantation.

Table 1 .
Characteristics of lung transplant recipient and infectious outcomes, n (%) 1IA high-risk conditions.2Number of patients negative in plasma samples but positive in BAL samples (minimum >3000 CMV IU/mL).